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Molecular structure of α, β, [delta] γ-tetraphenylporphinatoindium(III) chloride, and perturbed angular… Lee, Kai Mon 1979

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MOLECULAR STRUCTURE OF a,6,6,y-TETRA-PHENYLPORPHINATOINDIUM(III) CHLORIDE, AND PERTURBED ANGULAR CORRELATION STUDY ON RECONSTITUTED MYOGLOBIN by KAI MON,\LEE B. Tech. (Hons.), U n i v e r s i t y of Bradford, England, 1977, Grad RIC A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n CHEMISTRY THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1979 © K a i Mon, Lee, 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e . U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f , The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 - i i -ABSTRACT The f i r s t c r y s t a l and molecular structure of an indium porphyrin, a,$,6,Y-tetraphenylporphinatoindium(III) chloride (InTPP:Cl) has been determined by X-ray d i f f r a c t i o n . The indium i s five-coordinate i n a square-pyramidal complex with chloride as the a x i a l ligand. The o o average In-N bond i s 2.156(6)A, with an In-Cl distance of 2.369(2)A. The porphinato core i s somewhat expanded with an average radius of o o 2.067(3)A, and the macrocycle i s non-planar with a net doming (0.1A) o toward the indium. The indium atom i s located 0.61A above the mean plane of the four pyrrole nitrogen atoms. Based on the high degree of analogy between the p r i n c i p a l s t r u c t u r a l features of InTPP:Cl, Fe(III) porphyrins, and Fe(II) deoxyhemoglobin (e.g., displacement of metal atom above the mean porphyrin plane, distance from the metal to pyrrole nitrogen, porphyrin core expansion, doming of porphyrin s k e l e t a l core atoms), the present study suggests that r e c o n s t i t u t i o n of an indium porphyrin into apohemoglobin and apomyoglobin should produce the quaternary "tense" conformation. This provides an unique opportunity to study the porphyrin-apoprotein i n t e r a c t i o n s i n the "T" state of hemoglobin and myoglobin. The present r e c o n s t i t u t i o n of indium-Ill meso-protoporphyrin IX (^^InMPP IX) into myoglobin represents the f i r s t l o c a t i o n of a motional probe at the ce n t r a l metal of the act i v e s i t e of the protein. The r o t a t i o n a l c o r r e l a t i o n time (T c) of the reconstituted myoglobin has been determined by the Perturbed Angular Correlations (P.A.C.) technique. Since. ^ "^InMPP IX i s s t r u c t u r a l l y s i m i l a r to f e r r o -protoporphyrin IX (heme), there should be very l i t t l e i n t e r n a l r o t a t i o n - i i i -of the InMPP IX at the a c t i v e s i t e of the r e c o n s t i t u t e d myoglobin. Furthermore, the i n d i u m - l a b e l l e d myoglobin i s very l i k e l y to r e t a i n i t s n a t i v e conformation, i n c o n t r a s t to other l a b e l l i n g techniques i n which there i s always u n c e r t a i n t y as how much the l a b e l d i s t o r t s the s t r u c t u r e of the p r o t e i n being s t u d i e d . F i n a l l y , since no a d d i t i o n a l f l e x i b i l i t y i s introduced at the l a b e l l e d s i t e of the p r o t e i n , the present determination of T c(P.A.C.) f o r myoglobin i s more d i r e c t than fluorescence d e p o l a r i z a t i o n or ESR determinations. - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES v i i LIST OF TABLES x i ACKNOWLEDGEMENTS x i i CHAPTER I . GENERAL INTRODUCTION 1 Part One. C r y s t a l and Molecular S t r u c t u r e of 6 a, 3 , Y > <5-Tetraphenylporphinatoindium ( I I I ) C h l o r i d e CHAPTER I I . 7 2.1 M e t a l l o p o r p h y r i n Stereochemistry 7 2.1a Iron Porphyrins 18 2.1b Cobalt Porphyrins 21 2.2 A p p l i c a t i o n of M e t a l l o p o r p h y r i n 24 Stereochemistry to Heme Stereochemistry i n Hemoproteins (Myoglobin and Hemoglobin) 2.3 Myoglobin and Hemoglobin - The Heme 34 Group/Globin S t r u c t u r e 2.4 Models f o r Myoglobin and Hemoglobin 41 CHAPTER I I I . EXPERIMENTAL WORK 3.1 P r e p a r a t i o n of a ,3 ,y,(S-Tetraphenylporphinato- 45 indi u m ( I I I ) C h l o r i d e 3.2a P u r i f i c a t i o n of a,g,Yj<$-Tetraphenylporphinato- 48 indi u m ( I I I ) C h l o r i d e 3.2b T h i n - l a y e r Chromatography 48 3.2c V i s i b l e Absorption Spectroscopy 48 3.2d Nuclear Magnetic Resonance Spectroscopy 51 3.3 R e c r y s t a l l i s a t i o n of a,3 , y , 6-Tetraphenylporph- 51 in a t o i n d i u m ( I I I ) C h l o r i d e -V-Page CHAPTER IV. RESULTS AND DISCUSSION 4.1 Metal Displacements Above Mean Pyrrole Nitrogen Plane 4.2 Bond Lengths and Angles 4.3 Doming of the Porphyrin Skeleton 4.4 Phenyl Rings: Crystal Packing Part Two. Perturbed Angular Correlation Study on Myoglobin (Radioactive ^-In-labelled porphyrin/ CHAPTER V. Reconstituion/T c determination) PERTURBED ANGULAR CORRELATION GENERAL INTRODUCTION CHAPTER VI. THEORY OF PERTURBED ANGULAR CORRELATION OF NUCLEAR RADIATION 6.1 Introduction 6.2 Theoretical Consideration 6.2a Time-Dependent Quadrupole Interactions -The Limit of Rapid Motion 6.2b Static Electric Field Quadrupole Interactions - Polycrystalline Samples 6.2c Time-Dependent Quadrupole Interactions -The Limit of Slow Motion 6.2d Fields Without Axial Symmetry CHAPTER VII. APPLICATIONS OF GAMMA-GAMMA CORRELATIONS TO BIOLOGICAL MACROMOLECULES CHAPTER VIII.EXPERIMENTAL WORK 8.1 Preparation of Meso-Protoporphyrin IX 8.2 Preparation of Non-Radioactive Indium-III Meso-protoporphyrin IX 8.2a Thin-layer chromatography 52 52 61 62 65 75 76 79 79 82 90 95 99 106 111 118 118 123 124 -vi-Page 8.2b 8.2c 8.3 8.A 8.5 8.6 8.6a 8.6b 8.6c CHAPTER IX. 9.1 9.2 9.2a 9.2b 9.2c Appendix A. Appendix B. Appendix C. P u r i f i c a t i o n of Indium-III Meso-protoporphyrin IX V i s i b l e Absorption Spectroscopy P r e p a r a t i o n of Radioactive I n d i u m - I l l Meso-protoporphyrin IX Pre p a r a t i o n of Apo-rayoglobin S o l u t i o n R e c o n s t i t u t i o n of Apo-myoglobin w i t h I n d i u m - I l l Meso-proto-porphyrin IX Perturbed Angular C o r r e l a t i o n Measurements Detectors E l e c t r o n i c s Method RESULTS AND DISCUSSION Results - T i m e - D i f f e r e n t i a l Perturbed Angular C o r r e l a t i o n Data D i s c u s s i o n A n a l y s i s of the T i m e - D i f f e r e n t i a l Perturbed Angular C o r r e l a t i o n Spectra Least-Squares Analyses of the T i m e - D i f f e r e n t i a l Perturbed Angular C o r r e l a t i o n Data Comments on the R o t a t i o n a l C o r r e l a t i o n Times of Myoglobin determined by the Perturbed Angular C o r r e l a t i o n s Technique Co-operative E f f e c t of R e v e r s i b l e Oxygenation i n hemoglobin Tetraphenylporphine D i a c i d The E f f e c t of E x c i t a t i o n of the E l e c t r o n S h e l l on Angular C o r r e l a t i o n 123 125 128 130 132 135 135 136 138 141 141 149 149 151 153 164 167 169 References 172 - v i i -LIST OF FIGURES Figure T i t l e 2.1 The nucleus of porphine, i l l u s t r a t i n g the p o s i t i o n s of p o s s i b l e s u b s t i t u e n t s and the n o t a t i o n used f o r the unique atoms of the core 2.2 The p e r i o d i c t a b l e of metalloporphyrins 2.3 A diagram of the square-pyramidal c o o r d i n a t i o n group f o r f i v e - c o o r d i n a t e metalloporphyrins 2.A Energy-level diagram f o r square-pyramidal c o o r d i n a t i o n 2.5 C a l c u l a t i o n of the "doming" of the porphinato core 2.6 I l l u s t r a t i o n of c a l c u l a t i o n of the t o t a l movement of the proximal h i s t i d i n e i n hemoglobin and Co-hemoglobin 2.7 I l l u s t r a t i o n of p o s s i b l e p o i n t s of d i s t o r t i o n of Co(II) porphyrin p r o s t h e t i c group 2.8 Ferro-protoporphyrin IX (heme) and i t s coordinate system 2.9 Attachment of heme i r o n to N of h i s t i d i n e F8 2.10 A three-dimensional s t e r e o - p a i r drawing of myoglobin 2.11 The f o l d i n g and packing of chains i n hemoglobin 2.12 A schematic diagram p-oxo-bis (porphyriniron (III)) 2.13 " P i c k e t - f e n c e " porphyrin 2.1A "Capped" porphyrin 2.15 Chelated hemes having v a r y i n g degrees of s t e r i c hindrance toward base c h e l a t i o n 3.1 V i s i b l e a b s o r p t i o n spectrum of InTPP:Cl i n chloroform 3.2 V i s i b l e a b s o r p t i o n spectrum of ineso-TPP i n chloroform A . l Atomic numbering scheme f o r c r y s t a l l i n e InTPPrCl A.2 Stereoscopic view of the contents of one u n i t c e l l of c r y s t a l l i n e InTPPrCl - v i i i --ix-Figure Title Page 6.12 Differential .attentuation coefficients G22(0 107 for rhombic quadrupole interaction in poly-crystalline sources for intermediate state spins 1=2 and 1=5/2 6.13 Time-integrated attentuation coefficients 108 G22(°°) for rhombic quadrupole interaction in polycrystalline sources for spins 1=2 and 1=5/2 181 6.14 Differential anisotropy A(t) of the Ta y-y ] QO, directional correlation measured with a polycrystalline Hf-metal source 7.1 Anisotropy of correlated gamma-ray emission 112 from (a) free m C d + in solution (b) m C d + in the presence of native carbonic anhydrase (c) Hl mCd+ in the presence of apo-carbonic anhydrase 7.2 In vivo angular correlations of gamma-rays 115 following intravenous injection of three labelled indium compounds into mice 7.3 Time-differential perturbed angular correlation 117 spectra for samples of polyglutamic acid at pH=4.0 and pH=7.8 8.1 Visible Absorption spectrum of protoporphyrin "121 IX in formic acid 8.2 Visible absorption spectrum of meso-protoporphyrin 122 IX in formic acid 8.3 Tic results for indium meso-protoporphyrin IX 124 8.4 Visible absorption spectrum of indium meso-protoporphyrin IX in formic acid 8.5 Visible absorption spectrum of indium meso-protoporphyrin IX in benzene 8.6 A schematic drawing of a CM-52 column constructed from a 30cc plastic disposable syringe 126 127 134 8 . 7 A schematic diagram for a fast-slow arrangement -j -yj for P.A.C. experiments 8 . 8 A simplified block diagram of the electronics used in the present time-differential perturbed angular correlation experiments 139 - x -Figure T i t l e 9.1 TDPAC spectrum f o r ^ 1 1InMPP-Mb i n aqueous sodium phosphate b u f f e r (pH = 7.0) w i t h g l y c e r i n e added to give a f i n a l g l y c e r i n e percentage of A l . 9.2 TDPAC spectrum f o r *^InMPP-Mb i n aqueous sodium phosphate b u f f e r (pH = 7.0) without g l y c e r i n e 9.3 TDPAC spectrum f o r 1 1 1InMPP-Mb i n the l y o p h i l i z e d powder form 9.A TDPAC spectrum f o r ^^InMPP IX i n dimethylformamide 9.5 TDPAC spectrum f o r 1 1 1 I n T P P i n chloroform 9.6 R o t a t i o n a l c o r r e l a t i o n time f o r oxyhemoglobin as a f u n c t i o n of v i s c o s i t y A. l Oxygen e q u i l i b r i u m curves of myoglobin and hemoglobin B. l A schematic drawing of meso-tetraphenyl porphine ( f r e e base) B.2 A schematic drawing of a porphyrin d i a c i d B.3 Geometries f o r porphyrin d i a c i d s - x i -LIST OF TABLES Table T i t l e Page 2.1 Common porphyrins and abbreviations 7 2.2 Variations of M-N distances with ionic r a d i i 13 2.3 Stereochemical parameters for porphyrins 17 and metalloporphyrins 2.A Parameters of the square-pyramidal co- 20 ordination group i n several high-spin i r o n ( I I I ) porphyrins 2.5 Stereochemical parameters of the square- 22 pyramidal coordination group for low-spin c o b a l t ( I I ) porphyrins 2.6 Heme geometry i n myoglobin and hemoglobin 33 derivatives 4.1 Crystal data and conditions for data c o l l e c t i o n 62 4.2 F i n a l atomic p o s i t i o n a l and thermal parameters 68 4.3 Rigid group parameters 59 4.A Derived hydrogen atom p o s i t i o n a l and thermal 70 parameters A . 5 Selected interatomic distances and angles 71 A.6 Selected planes of the porphyrin macrocyclic 73 skeleton A.7 A compilation of metalloporphyrins with metal 74 out-of-plane displacements 7.1 Anisotropies for In compounds studied ^n vivo 9.1 F i t t e d values for T c,n, w D and 6 for myoglobin 143 reconstituted wi th -L-L1InMPP IX. 9.2 Rotational c o r r e l a t i o n times, i c , and Stokes 156 radius (R), of myoglobin, chymotrypsin, and hemoglobins 9.3 Rotational c o r r e l a t i o n times, T c of myoglobin 158 and myoglobin reconstituted with minMPP IX at various temperatures and v i s c o s i t i e s 9.A Stokes radius of 1 1 1InMPP-Mb calculated from 162 the Debye model of relaxation using experimental T (P.A.C.) values - x i i -ACKNOWLEDGEMENTS In 1977, I j o i n e d Prof. A. G. Marshall's b i o p h y s i c a l group i n U.B.C. where I was f i r s t introduced to the Perturbed Angular C o r r e l a t i o n (P.A.C.) of gamma r a d i a t i o n technique, and porphyrin chemistry. I should l i k e to express my s i n c e r e a p p r e c i a t i o n to Prof. M a r s h a l l f o r h i s c o n t i n u i n g guidance and encouragement throughout my stay at U.B.C. I am p a r t i c u l a r l y indebted to Prof. P.W. M a r t i n of the Physics Department, U.B.C. who c a r r i e d out the P.A.C. measurements, and a l s o to Dr. R. G. B a l l who d i d the X-ray a n a l y s i s of the indium porphyrin complex. A s p e c i a l thanks to Miss Mandy of the Pathology Department, U.B.C. who c a r r i e d out the v i s c o s i t y measurements. I wish to acknowledge w i t h g r a t i t u d e the h e l p f u l and c r i t i c a l suggestions from Drs. D. C. Roe, Lewis Choi, Mr. G. A. Louma and J . L. Smith who read t h i s t h e s i s . A measure of acknowledgement must be extended to Mr. T. Dekleva, R. Snoek, Ms. Roxanne LeBlanc Lemieux, and Mrs. J . Carruthers f o r t h e i r support and encouragement. I would l i k e to thank Dr. A. W. Addison of Drexel U n i v e r s i t y , and Drs. D. Dolphin, B. R. James, A. S t o r r and J . T r o t t e r of U.B.C. f o r t h e i r h e l p f u l comments and d i s c u s s i o n s . Although space l i m i t a t i o n s p r o h i b i t a l i s t i n g of a l l those who have make my stay an enjoyable and f r u i t f u l one i n Vancouver, I wish to record my g r a t i t u t e to them. F i n a l l y , I would l i k e to express my ap p r e c i a t i o n to Mrs. Anna Wong f o r t y p i n g t h i s t h e s i s . -1-CHAPTER I GENERAL INTRODUCTION The l a s t decade has witnessed a number of major p u b l i c a t i o n s i n metalloporphyrin research. A high l e v e l of con t i n u i n g a c t i v i t y i n t h i s f i e l d stems from i n t e r e s t i n b i o l o g i c a l systems to which these compounds are r e l a t e d . Metalloporphyrins are a l s o s t u d i e d f o r developing super-conductors ( 1 ) , anti-cancer drugs ( 2 ) , and c a t a l y s t s (3). The b i o l o g i c a l l y important metalloporphyrins are p r i n c i p a l l y . i r o n porphyrins, and these are the porphyrins that serve as the p r o s t h e t i c group i n s e v e r a l c l a s s e s of the hemoproteins (4) such as hemoglobins, myoglobins, c a t a l a s e s , e r y t h r o c r u o r i n s , some peroxidases, and cytochromes. For the present case, only myoglobins and hemoglobins w i l l be considered. This t h e s i s i s d i v i d e d i n t o two p a r t s . Part one deals w i t h the stereochemistry of indiu m ( I I I ) meso-tetraphenylporphyrin c h l o r i d e (InTPPrCl) and metalloporphyrins i n general. Part two describes the deter-mination of the r o t a t i o n a l c o r r e l a t i o n time of r e c o n s t i t u t e d myoglobin by means of the d i r e c t i o n a l gamma-gamma angular c o r r e l a t i o n technique. S t r u c t u r a l s t u d i e s of metalloporphyrins c o n t r i b u t e s i g n i f i c a n t l y to the understanding of the chemical and p h y s i c a l p r o p e r t i e s of porphyrins, and e l u c i d a t i o n of the s t r u c t u r e of i r o n porphyrins i n p a r t i c u l a r c a r r i e s many b i o l o g i c a l l y s i g n i f i c a n t i m p l i c a t i o n s ; t h i s aspect i s d e a l t w i t h i n d e t a i l s i n Chapter I. According to the most recent published c o m p i l a t i o n ( 5 ) , the c r y s t a l and molecular s t r u c t u r e s of more than 120 porphyrins and metalloporphyrins had been determined by X-ray d i f f r a c t i o n by 1976. A major reason f o r the -2-high and co n t i n u i n g i n t e r e s t i n such d e t a i l s as the bond l e n g t h s , metal d i s p o s i t i o n , and d i s t o r t i o n s i n the porphyrin core s t r u c t u r e of m e t a l l o -porphyrins i s that pronounced v a r i a t i o n s i n these s t r u c t u r a l parameters occur on oxygen-binding, and these v a r i a t i o n s are proposed to r e l a t e d i r e c t l y to the co-operative oxygen-binding f u n c t i o n of hemoglobin (6). Although at l e a s t 20 met a l l o p o r p h y r i n complexes e x h i b i t s t r u c t u r e s w i t h the metal atom d i s p l a c e d by v a r y i n g distances above the mean plane of the 4 p y r r o l e nitrogens of the porphyrin s k e l e t o n , v i r t u a l l y none f u l f i l l s the u s e f u l requirements that the metal displacement be s i m i l a r to that i n hemoglobin, and that the metal be s t a b l e to o x i d a t i o n . Since i n d i u m ( I I I ) i s o x i d a t i o n - s t a b l e i n aqueous s o l u t i o n , and has an i o n i c r a d i u s s i m i l i a r to that of i r o n ( I I ) , i t was decided to determine the c r y s t a l and molecular s t r u c t u r e of an indium porphyrin complex. The present r e s u l t s suggest that r e c o n s t i t u t i o n of myoglobin or hemoglobin w i t h indium porphyrins should provide i n f o r m a t i o n of d i r e c t relevance to the proposed models f o r hemoglobin f u n c t i o n . The p o t e n t i a l of perturbed angular c o r r e l a t i o n (P.A.C.) measure-ments to y i e l d motional and s t r u c t u r a l i n f o r m a t i o n about b i o l o g i c a l macromolecules has been discussed i n s e v e r a l p u b l i c a t i o n s (7-12). The s i m p l i c i t y of experimental measurements, i n combination w i t h c o n c e n t r a t i o n -12 s e n s i t i v i t y approaching 10 M enhance the appeal of the P.A.C. technique. Another a t t r a c t i v e f e a t u r e of the P.A.C. technique i s the a b i l i t y to study both the l i q u i d and s o l i d s t a t e s , which opens the p o s s i b i l i t y f o r " i n v i v o " experimentation. Despite the obvious p o t e n t i a l of such a p p l i c a t i o n s , r e l a t i v e l y few s t u d i e s of t h i s type have been performed. The reason f o r t h i s p a u c i t y of p u b l i c a t i o n s i s probably the l a c k of v e r s a t i l i t y i n s e l e c t i v e attachment of r a d i o a c t i v e " r o t a t i o n a l labels," to s p e c i f i c s i t e s on macromolecules. This t h e s i s shows that i n d i u m - I l l -3-l a b e l l e d porphyrins can be s e l e c t i v e l y incorporated i n t o myoglobin. Indium has an isotope ("'""'""'"In) that i s r a d i o a c t i v e w i t h an i n i t i a l s t a t e h a l f - l i f e of 2.8 days, and the decay process produces two gamma rays i n succession, each w i t h a convenient energy f o r d e t e c t i o n . M a r s h a l l et a l . , (13,14) were the f i r s t to show how gamma-gamma c o i n -cidence measurements f o r t h i s s o r t of energy cascade can give d i r e c t i n f o r m a t i o n about the chemical bonding and motional f l e x i b i l i t y at an ind i u m - l a b e l l e d s i t e on a macromolecule. The present r e s u l t s (Part two) confirm the a p p l i c a b i l i t y of t h i s measurement technique to i n d i u m - l a b e l l e d myoglobin. The present r e c o n s t i t u t i o n of indium-III meso-tetraphenylporphyrin i n t o myoglobin represents the f i r s t motional probe l o c a t e d at the c e n t r a l metal at the center of the a c t i v e s i t e of p r o t e i n . J u s t i f i c a t i o n f o r the content of t h i s t h e s i s i s two-fold. F i r s t l y , .this t h e s i s proposes that r e c o n s t i t u t i o n . of hemoglobin w i t h indium porphyrins could provide an i n s i g h t i n t o the mechanism of co-operative oxygen-binding f u n c t i o n of hemoglobin. Secondly, the present work demonstrates that the P.A.C. technique could provide an a l t e r n a t i v e means of o b t a i n i n g r o t a t i o n a l c o r r e l a t i o n times of b i o l o g i c a l macromolecules. Hemoglobin i s p o s t u l a t e d by Perutz (6) to e x i s t i n two conformational forms. The form i n which the c e n t r a l f i v e - c o o r d i n a t e F e ( I I ) atom i s d i s -placed above the heme plane i s known as the "T" ("tense") form. The other "R" ("relaxed") form i s one i n which the c e n t r a l s i x - c o o r d i n a t e F e ( I I ) atom l i e s n e a r l y coplanar w i t h the heme plane. The heme i r o n i s f i v e -coordinate i n deoxy-hemoglobin (deoxy-Hb) which i s r e s p o n s i b l e f o r the "T" form. On bi n d i n g molecular oxygen, the heme i r o n becomes s i x - c o o r d i n a t e and moves i n t o the heme plane, and the oxy-Hb i s s a i d to be i n the "R" form. -4-According to Pe r u t z , the t r a n s i t i o n from "T" to "R" forms causes p r o t e i n conformational changes that are necessary f o r the observed co-operative oxygenation e f f e c t i n hemoglobin. A c r i t i c a l t e s t .for t h i s proposal would be to " f r e e z e " one or more subunits i n t o the "T" form, and then monitor changes i n the k i n e t i c , e l e c t r o n i c , and motional p r o p e r t i e s of the remaining n a t i v e subunits upon oxygenation. In oxy-Hb, n a t i v e sub-u n i t s i n the"T" forms are very d i f f i c u l t to o b t a i n s i n c e F e ( I I ) binds oxygen to give the "R" form. Since the "T" form cannot be s t a b l y maintained i n oxy-Hb using n a t i v e i r o n heme, other means such as r e c o n s t i t u t i o n or "metal r e p l a c e -ment" must be sought. Hemoglobins have been r e c o n s t i t u t e d w i t h v a r i o u s metalloporphyrins c o n t a i n i n g Mn, Co, Zn, and Mg i n an o x i d a t i o n s t a t e s i m i l a r to F e ( I I ) . However, Co binds oxygen, and Mn(II) a u t o x i d i z e s to Mn ( I I I ) . Both Mg and Zn may be s t a b l e to o x i d a t i o n but they e x h i b i t r e l a t i v e l y s m a ll out-of-plane displacements of the metal atoms above the porphyrin plane r e l a t i v e to that of the Fe atom i n n a t i v e Hb. A comparison of the s t r u c t u r a l data on various metalloporphyrins (Table 4.7) shows that the out-of-plane displacement of indium i n InTPP:Cl i s by f a r the most s i m i l a r to that of Fe i n horse deoxy-Hb and human deoxy-Hb. Other a t t r a c t i v e features of indium are that I n ( I I I ) i s o x i d a t i o n s t a b l e , and does not bind oxygen. The combination of n e a r - i d e a l In...Pu* d i s t a n c e , s t a b i l i t y to o x i d a t i o n , and i n a b i l i t y to bind oxygen suggest that an indium porphyrin may be an i d e a l candidate f o r inducing the "T" conformation. I f the current theory of c o - o p e r a t i v i t y by Perutz (6) i s c o r r e c t , indium porphyrin Indium out-of-plane displacement from the mean plane of the e n t i r e macrocycle. -5-complexes r e c o n s t i t u t e d i n place of heme groups i n hemoglobin should " f r e e z e " the i n d i u m - l a b e l l e d subunits i n t o the d e s i r e d "T" conformation. The second j u s t i f i c a t i o n i s that P.A.C. technique can be used to ob t a i n motional and s t r u c t u r a l i n f o r m a t i o n about b i o l o g i c a l macromolecules. Indium has an advantage that i t has an isotope ("'""'""'"In) that i s s u i t a b l e f o r gamma-gamma coincidence measurements. These measurements r e q u i r e the i n c o r p o r a t i o n of a gamma-ray emitter i n t o the molecule under study. Several isotopes can be used: ^"'""'"mCd(tj =49 min), ^ Z n ( t j = 9 h ) , ^ ^ H g ( t j =43 min), -5 -3 -i "PbCt, =68 min), and In(tj=2.8 days). I t i s obvious that In i s the most convenient isotope from-the p r a c t i c a l viewpoint. The present r e s u l t s e f f e c t i v e l y demonstrate that motional p r o p e r t i e s such as the r o t a t i o n a l c o r r e l a t i o n time of myoglobin can be determined by means of the P.A.C. technique. This work can be extended to other b i o l o g i c a l macromolecules such hemoglobins, and cytochromes. I t i s i n t e r e s t i n g to note that f o r the past decades, many i n v e s t i -gators have u t i l i z e d v a r i o us marker substances ranging from porphyrin complexes, t e t r a c y c l i n e d e r i v a t i v e s , r a d i o a c t i v e i s o t o p e s , and other substances to i d e n t i f y and d e l i n e a t e malignant t i s s u e s . Schwartz and coworkers have observed a high concentration of c e r t a i n porphyrins i n tumors. I t i s conceivable that "'""'""'"In-labelled porphyrins could be used f o r t u m o r - l o c a l i s a t i o n . I n d i u m - I l l has i d e a l p h y s i c a l c h a r a c t e r i s t i c s (a convenient 2.8 days h a l f - l i f e and gamma rays i n the d e s i r e d energy range) f o r tumor scanning. -6-PART ONE CRYSTAL AND MOLECULAR STRUCTURE OF a , 3,Y , < 5 -TETRAPHENYLPORPHINATOINDIUM(III) CHLORIDE -7-CHAPTER I I 2.1 M e t a l l o p o r p h y r i n Stereochemistry The porphyrins are compounds formed by adding s u b s t i t u e n t s to the nucleus of porphine (Figure 2.1). The n a t u r a l l y o c c u r r i n g porphyrins are g e n e r a l l y formed by adding s u b s t i t u e n t s to p o s i t i o n s 1-8 and are named according to the number and type of s u b s t i t u e n t s . Table 2.1 l i s t s s e v e r a l porphyrins, both n a t u r a l and s y n t h e t i c w i t h t h e i r t r i v i a l names that most f r e q u e n t l y employed f o r s t r u c t u r a l s t u d i e s . TABLE 2.1 Common Porphyrins and Abbreviations  . . . a P o s i t i o n on r i n g Porphyrin 1 2 3 4 5 6 7 8 a ,3,6,y M V M V M P P M. H M Ac M Ac M P P M H M E M E M P P M H M E M E M E M E H M E E M M E E M H E E E E E E E E H H H H H H H H H C 6 H 5 H H H H H H H H C 5H 4N Protoporphyrin 2,4-Diacetyldeutero-p o r p h y r i n c Mesoporphyrin^ E t i o p o r p h y r i n I e E t i o p o r p h y r i n 11^ Octaethylporphyring T e t r a p h e n y l p o r p h y r i n n T e t r a ( 4 - p y r i d y l ) -p o r p h y r i n 1 Ac,COCH3;E,CH2CH3;M,CH3;P,CH2CH2COOH;V,CHCH2 H 2Proto. H 2Deut. H2MesoP. "H 2Etio I. H 2 E t i o I I . g H2OEP. hH 2TPP. H 2TPyP. The n o t a t i o n that w i l l be used throughout t h i s chapter i s i l l u s t r a t e d -8-i n F i g ure 2.1. and f o r a- and b-carbon atoms of a p y r r o l e r i n g , N f o r p y r r o l e n i t r o g e n , and Cm f o r methine carbon. Ct denotes the center of the porphyrin, M a coordinated metal i o n , and Ne the co-ordinated a x i a l nitrogeneous base. M-N i s used to represent a metal-p y r r o l e n i t r o g e n bond d i s t a n c e , and M-Ne the m e t a l - a x i a l n i t r o g e n bond dist a n c e . A non-bonded separation between two atoms i s denoted by three centered d o t s ( . . . ) ; a bonded di s t a n c e i s designated by a dash(-). The c h i e f impediment to the e l u c i d a t i o n of met a l l o p o r p h y r i n s t r u c t u r e i s the d i f f i c u l t y ( e s p e c i a l l y f o r d e r i v a t i v e s of b i o l o g i c a l l y important protoporphyrin IX) of o b t a i n i n g the s i n g l e c r y s t a l s s u i t a b l e f o r X-ray d i f f r a c t i o n a n a l y s i s . The c r y s t a l s are o f t e n too sm a l l or when l a r g e enough, are o f t e n subject to c r y s t a l defect due to i n t e r n a l packing d i s o r d e r . The nature of the c r y s t a l d i s o r d e r may vary markedly both i n kind and degree. I t i s o f t e n found that c r y s t a l l i n e d i s o r d e r can be so extensive so as to preclude meaningful s t r u c t u r a l i n v e s t i g a t i o n . S t r u c t u r a l a n a l y s i s can o f t e n be misleading i f the nature of the c r y -s t a l l i n e defect i s not i d e n t i f i e d and i t s importance assessed. For example, the present c r y s t a l l o g r a p h i c a n a l y s i s of InTPP:Cl i n d i c a t e s non-equivalence of the phenyl t i l t angles which can be explained from i n t e r n a l packing. D e r i v a t i v e s of the symmetric porphyrins (H^EtiOjR^OEP, H^TPP, and R^TPyP) are most f r e q u e n t l y employed f o r s t r u c t u r a l determination because of t h e i r a v a i l a b i l i t y and ease i n o b t a i n i n g the necessary s i n g l e c r y s t a l s . W i t h i n the accuracy of the experimental data, there are only small d i f f e r e n c e s between the n a t u r a l l y o c c u r r i n g porphyrins and s y n t h e t i c d e r i v a t i v e s , i n cases where s t r u c t u r e s of. both have been determined. Removal of the two p y r r o l e protons y i e l d s a porphyrin d i a n i o n that -9-R 5 Re R 4 Figure 2.1. The nucleus of porphine, I l lustrating the positions of possible subst i tuents and the notation used for the unique atoms of the core. -10-behaves as a t e t r a d e n t a t e l i g a n d which r e a d i l y complexes any of a v a r i e t y of metal ions. As expected, the minimum c o o r d i n a t i o n number i s f our. A d d i t i o n of a x i a l l i g a n d s , e i t h e r n e u t r a l or a n i o n i c , gives metalloporphyrin d e r i v a t i v e s wherein the metal i o n has a c o o r d i n a t i o n of f i v e , s i x or e i g h t . Only the f i v e - c o o r d i n a t e metalloporphyrins are of i n t e r e s t i n the present case. F i g u r e 2.2 gives the " p e r i o d i c -t a b l e of metalloporphyrins". Porphyrins, i n common w i t h other macrocyclic l i g a n d s , have a c e n t r a l hole of e s s e n t i a l l y f i x e d dimensions. The porphinato core r e s i s t s undue r a d i a l expansion and c o n t r a c t i o n i n e q u a t o r i a l plane. In c e r t a i n metal complexes, the metal i s unable to f i t i n t o t h i s hole and, as has been shown by Hoard (15). Thus, the perpendicular displacement of the metal atom i s r e l a t e d to the e f f e c t i v e r a d i u s , s i n c e the s i z e of the c e n t r a l hole i n the porphyrin i s r e l a t i v e l y constant. An i n s p e c t i o n of Table 4.7 shows no simple c o r r e l a t i o n between i o n i c , r a d i i and metal i o n d i s p l a c e -ments. The porphyrins are not r i g i d but r a t h e r remarkably f l e x i b l e macrocycle molecules (16,17,18). In 1961 R.J.P. Wil l i a m s (19) pointed out that the t r a n s i t i o n from hi g h to low s p i n should be accompanied by a marked r e d u c t i o n i n the i o n i c r a d i u s of the i r o n . He f u r t h e r suggested that change i n the s t r u c t u r e of hemoglobin on oxygenation " i s r e l a t e d to the change i n the i m i d a z o l e -i r o n d i s t a n c e or p o s s i b l y to the concomitant change i n the s t e r i c arrange-ment of the imidazole (of the heme-linked h i s t i d i n e ) r e l a t i v e to the porphyrin plane". From the c r y s t a l l o g r a p h i c s t u d i e s of metalloporphyrins ( e s p e c i a l l y f e r r i c porphyrin complexes), J . L. Hoard and a s s o c i a t e s demonstrated Li B Na Mg» Al Si P K Ca Sc Ti V Cr M n ' F e * Co* Ni* Cu» Zn» G a G e A s R b Sr Y Z r Nb Mo Tc R u R h P d A g C d In Sn S b Cs Ba La H f Ta W R e Os I r P t A u H g Tl P b Bi . . P r . . E u . . Y b . . Th Figure 2.2. The periodic table of metalloporphyrins.(Metals which are inserted by nature are marked with an asterisk.) -12-e x p e r i m e n t a l l y t h a t t h e h i g h t o low s p i n t r a n s i t i o n i s accompanied by a marked r e d u c t i o n i n r a d i u s of t h e i r o n (15,20,21,22,23). He p r e d i c t e d t h a t t h e same s h o u l d h o l d t r u e f o r f e r r o u s complexes., Hoard t h e n p r o p o s e d t h a t t h e : h i g h - s p i n f e r r o u s i o n i n deoxy-Hb and deoxy-Mb i s f i v e -c o o r d i n a t e d and i s d i s p l a c e d from t h e p o r p h y r i n p l a n e toward t h e p r o x i m a l o h i s t i d i n e by 0.5-0.8A. Upon b i n d i n g an oxygen m o l e c u l e , , t h e i r o n atom becomes s i x - c o o r d i n a t e and l o w - s p i n , and moves back toward t h e p l a n e o f p o r p h y r i n . T h i s p l a n a r i t y has been v e r i f i e d i n cyanomethemoglobin by H e n d r i c k s o n and Love ( 2 4 ) , and C O - e r y t h r o c u o r i n by .Huber e t a l , , ( 2 5 ) , and v e r y r e c e n t l y f o r o x y - m y o g l o b i n by S . P h i l l i p s ( 2 6 ) . W i l l i a m s ( 1 9 ) , and Hoard (27) s u g g e s t e d t h a t t h e movement of t h e i r o n atom causes a change i n t h e p o s i t i o n o f t h e i m i d a z o l e group o f t h e p r o x i m a l h i s t i d i n e . T h i s movement i s i n t u r n r e s p o n s i b l e f o r t h e c o n -f o r m a t i o n a l changes i n h e m o g l o b i n and m y o g l o b i n upon o x y g e n a t i o n . Based on h i s s t r u c t u r a l s t u d i e s of o x y ( m e t ) - and deoxy-Hb, P e r u t z (28,29) has p r o p o s e d t h e s t e r e o c h e m i c a l mechanism f o r t h e c o - o p e r a t i v e phenomenon (See A p p e n d i x A ) . A t t h e . h e a r t o f t h i s p r o p o s a l i s t h e a s s u m p t i o n , based on e a r l i e r i d e a s o f Hoard (.27)., and W i l l i a m s (19) t h a t i n h e m o g l o b i n t h e h i g h - s p i n t o l o w - s p i n t r a n s i t i o n s h o u l d be a s s o c i a t e d w i t h a movement o f t h e i r o n toward the. p o r p h y r i n p l a n e (22,23,27,28,30, 0 3 1 ) . The r e s u l t i n g m o t i o n of t h e p r o x i m a l h i s t i d i n e (>0.5A) c a u s e s t h e n e c e s s a r y c o n f o r m a t i o n a l changes i n t h e p r o t e i n t h a t a r e r e s p o n s i b l e f o r t h e c o - o p e r a t i v i t y . O b v i o u s l y , t h e p r i n c i p a l m e t a l l o p o r p h y r i n s t r u c t u r a l p a r a m e t e r s o f g r e a -t e s t i n t e r e s t a r e t h u s t h e d i s p l a c e m e n t o f t h e m e t a l atom from t h e mean p l a n e , Ct...M, and t h e M-N bond d i s t a n c e s . -13-The bond distances within the porphine skeleton i t s e l f are quite invariant with respect to the porphyrin compounds studied. As expected . the small distance v a r i a t i o n s are due to substituent e f f e c t s . The M-N bond distance, however, does vary appreciably depending on the nature of o the metal atoms coordinated to the porphyrin. M-N can range from 2.32A o i n bismuth porphyrins to 1.95A i n n i c k e l porphyrins. A long M-N bond can be expected i f the metal i s positioned out of the mean plane of the pyrrole nitrogen atoms. Table 2.2 shows that there i s no simple c o r r e l a t i o n between io n i c r a d i i and M-N distances. TABLE 2.2 Variations of M-N distances with i o n i c r a d i i Metal Ionic radius M-N distance o o Fe(III) 0.64A 2.07A Ni(II) 0.68 1.96 Cu(II) 0.72 1.98 Zn(II) 0.74 2.05 In(III) 0.81 2.16 Pd(II) 0.86 2.00 T l ( I I I ) 0.95 2.21 B i ( I I I ) 0.96 2.32 The porphinato core i s often not planar but rather e a s i l y subjected to deformation normal to the mean plane into a r u f f l e d or domed configuration. -14-Thls observation has been confirmed by s t r u c t u r a l determination of tetraphenylporphine (32) and i t s copper and palladium d e r i v a t i v e s (33). The InTPPrCl a l s o e x h i b i t s marked d e v i a t i o n s from p l a n a r i t y of the porphinato core (See Chapter IV, S e c t i o n 4.3). Planar conformations are necessary to ensure maximum i r - i n t e r a c t i o n between the s u b s t i t u e n t s and the extensive conjugated porphyrin systems. Nonplanar conformations, however, predominate i n c r y s t a l l i n e m etalloporphyrins. The l i k e l y ex-p l a n a t i o n i s that nonplanar conformations r e s u l t from packing c o n s t r a i n t s i n the c r y s t a l or i n t r a m o l e c u l a r r e p u l s i o n s of a x i a l l i g a n d s w i t h atoms of the core. Hoard (34) examined the c o r r e l a t i o n between M-N d i s t a n c e s and geometry of the macrocycle i n metalloporphyrins. He concludes that deformations are more l i k e l y f o r contracted core which has short M-N d i s t a n c e s . Recent stu d i e s (35,36) confirm Hoard's observations. C u l l e n and co-workers (35) compared two c r y s t a l l i n e forms of NiOEP so.as to e l i m i n a t e the e f f e c t s of d i f f e r e n t s u b s t i t u e n t s on the r i n g on the geometry. The t e t r a g o n a l form o has the average Ni-N d i s t a n c e (1.93A), the s h o r t e s t M-N d i s t a n c e yet r e -ported, f o r a m e t a l l o p o r p h y r i n , and the macrocycle i s h i g h l y d i s t o r t e d (37). In c o n t r a s t , the t r i c l i n i c . NiOEP.is e s s e n t i a l l y planar and the M-N d i s t a n c e o o i s 1.96A. The M-N d i s t a n c e of 2.01A appears to be a nearly optimal value of minimal s t r a i n and u n d i s t o r t e d accomodation of the metal atom w i t h i n the c e n t r a l porphinato core (21). S t r u c t u r a l s t u d i e s a l s o show that f o r a h i g h l y expanded core, the most e f f i c i e n t nonplanar conformation i s doming of the core (18,34). The present study of InTPPrCl a l s o confirms t h i s observation. InTPPrCl has a h i g h l y expanded porphinato core w i t h (M-N)^ o d i s t a n c e of 2.16A. As expected the plane of the four p y r r o l e nitrogens i s -15-d i s p l a c e d i . e . domed upward by 0.1A from the mean plane of the porphinato core. The f i v e - c o o r d i n a t e geometry i s a very common one i n met a l l o p o r p h y r i n s , e s p e c i a l l y prominent f o r z i n c , magnesium, c o b a l t ( I I ) , and h i g h - s p i n i r o n porphyrins. Figure 2.3 represents the schematic diagram of the square-pyramidal c o o r d i n a t i o n group f o r f i v e - c o o r d i n a t e m e t a l l o p o r p h y r i n s . A b r i e f c o n s i d e r a t i o n of the Pythagorean theorem demonstrates that the out-df-plane displacement, Ct...M of the metal atom from the mean porphinato core can be c a l c u l a t e d i f Ct...N and M-N dista n c e s are known. A modest doming of the core i s a ne a r l y i n v a r i a b l e f e a t u r e among f i v e - c o o r d i n a t e metalloporphyrins e s p e c i a l l y those w i t h l a r g e c e n t r a l metal atoms. Con-sequently, the doming of the core toward the metal atom leads to the i n e q u a l i t y of M...P„ < M...P . Generally P>T...P (doming) i s small N c N c ° o <0.05A. For t h i s c l a s s of met a l l o p o r p h y r i n s , t y p i c a l Ct...N values are o found i n the range of 1.98-2.05A, and the M-N distances are u s u a l l y o <2.01A (Table 2.3 and 4.7). The displacement of the metal atom from the mean plane i s a measurable property of a l l metalloporphyrins of o o t h i s c l a s s . The magnitude v a r i e s from 0.10A to >0.50A. The length of the a x i a l ligand-metal bond i s dependent on the nature of the a x i a l l i g a n d . Due to the doming e f f e c t , s t e r i c i n t e r a c t i o n between the a x i a l l i g a n d and the porphinato core i s reduced and l e s s important i n f i v e -coordinate than s i x - c o o r d i n a t e m e t a l l o p o r p h y r i n s ; i t s importance i s obviously diminished w i t h an i n c r e a s i n g displacement of the metal atom out-of-plane. A short d i s c u s s i o n of the stereochemistry of i r o n porphyrins i s -16-Figure 2.3. A diagram of the square -pyramida l coordination group for f ive-coordinate metalloporphyrins -17-warranted i n view of t h e i r r e l a t i o n s h i p s to b i o l o g i c a l l y important molecules such as hemoglobin and myoglobin. Cobalt porphyrins w i l l a l s o be included because of the r e l a t i o n of cobalt porphyrin s t e r e o -chemistry to the c o b a l t - s u b s t i t u t e d hemoproteins. A number of i n t e r e s t i n g features emerge from the stereochemical data i n Table 2.3. The metal atom l i e s i n plane w i t h the four p y r r o l e o n i t r o g e n atoms only i f M-N < 2.01A. The normal r a d i u s of the c e n t r a l "hole" i n an u n d i s t o r t e d metalloporphyrin has been estimated to be o o 2.01A (21). I t i s i n t e r e s t i n g to note that.the value of 2.01A c o r r e s -o ponds ne a r l y to the mean of the smallest (1.93A f o r t e t r a g o n a l N10EP) and o the l a r g e s t (2.21A f o r Tl(OEP)Cl) M-N values.(See Table. 4.7) so f a r reported f o r metalloporphyrins. The Table 2.3 a l s o shows that the metal i on d i s -placement i s s u b s t a n t i a l i n each of the i r o n porphyrins; a l l of these ( i n c l u d i n g f e r r i m y o g l o b i n) are h i g h - s p i n Fe d e r i v a t i v e s w i t h the exception of 2-MeImFe(II)TPP i n which i r o n atom i s i n o x i d a t i o n s t a t e two. „ n TABLE 2.3 Stereochemical parameters f o r p o r p ^ r i n s ^ a n d metalloporphyrins (distances M-Na Ct-N b A ° A x i a l . L i g a n d s Ref N i E t i o 1.957 1.957. None 38 NiDeut 1.960 1.960 - None 39 CuTPP 1.981 1.981 - None 33 PdTPP 2.009 2.009 - None 0 33 H20ZnTPP >2.05 2.042 >0.19 H 20 at < 2.21 A 20 ClFeTPP 2.049 2.012 0.383 C l at 2.19 A o 40 Chlorohemin 2.062 2.008 0.475 C l at 2.218 A OMe at 1.842 A 41 MeOFeMeso 2.073 2.022 0.455 23 Porphine - 2.051 - None 42 tet-TPP - 2.054 - None 17 tr-TPP - 2.065 - None 43 Fe(III)Mb -1.9 -1.9 -0.30 N at -1.9 0 44 H 20 ~2.1 A Metal- n i t r o g e n d i s t a n c e Radius of c e n t r a l hole: see t e x t Out-of-plane displacement of the metal atom From J . L. Hoard (Ref. 22) -18-2.1a Iron Porphyrins Iron porphyrins provide the t y p i c a l examples of the influence of the spin-states and oxidation states of the c e n t r a l metal ions on the stereochemistry of metalloporphyrin. The e f f e c t i v e radius of high-spin i r o n ( I I I ) ion i s too large to allow the ir o n to f i t into the ce n t r a l hole. o A t y p i c a l displacement of the ir o n of about 0.48A out-of-plane i s as expected. A small amount of doming of the porphinato core toward the ir o n ( I I ) o ion i s frequently observed with P c >..P N < 0.05A. A q u a l i t a t i v e p i c t ure for t h i s observation i s gained by considering a square-pyramidal model for a high-spin i r o n porphyrin. In the simple model, the Z-direction i s defined as normal to the porphinato core (Figure 2.4). The d: 2 o r b i t a l of a coordinated x y metal ion i s then i n a plane p a r a l l e l to the mean porphinato core. A metal ion where the d 2_ 2 o r b i t a l i s populated w i l l be r e l a t i v e l y large x y and have commensurately long M-N bonds. In any high-spin Fe(II) or Fe(III) metalloporphyrin, there i s one unpaired electron i n each of the 3d^ 2 y2 and 3d z2 o r b i t a l s i n the metal atom. The presence of the unpaired electron i n 3d^2_y2 o r b i t a l i s responsible f o r the sub s t a n t i a l displacement of the iro n from the plane of the pyrrole nitrogen atoms. In a low-spin Fe(II) or F e ( I I I ) , the p a i r i n g of electron spins i n the d ', d, , and d „ valence-6 r xy yz xz s h e l l o r b i t a l s of the metal atom allows f u l l u t i l i z a t i o n of the unoccupied 3d^2_y2 a n d 3 <^ z2 o r b i t a l s f o r complexing two a x i a l ligands. The high-spin complex of 2-methylimidazoleiron(II) (45) i s a f i v e -o coordinate, square-pyramidal, and the iro n ( I I ) i s displaced 0.42A from o the mean plane of the four pyrrole nitrogens and 0.55A from the mean o porphinato core. The doming i . e . P^...P separation i s 0.13A. -19-Figure 2 .4 . E n e r g y - l e v e l diagram for square -py ramida l coord inat ion . -20-Both the F e ( I I ) and F e ( I I I ) can form low-spin complexes when coordinated to two appropriate type of a x i a l l i g a n d s . According to Hoard's observation (15,20,21,23) i n the low-spin s i x - c o o r d i n a t e complexes, the i r o n i s coplanar or ne a r l y eoplanar w i t h the porphinato core. A s u b s t a n t i a l l y shorter Fe-N bond d i s t a n c e r e l a t i v e to the hi g h -s p i n can a l s o be a n t i c i p a t e d . The s t r u c t u r e of a low-spin s i x - c o o r d i n a t e ( I n ^ F e T P P ^ C l has shown o that the p o s i t i o n of the i r o n ( I I I ) i s only 0.009A (30) from the mean o porphinato n i t r o g e n s . The (Fe-N). d i s t a n c e of 1.989A i s s i g n i f i c a n t l y av o shorter than those of the h i g h - s p i n complexes w i t h ( F e - N ) a v > 2.04A (See Table 2.4). TABLE 2.4 Parameters of the Square-Pyramidal Coordination Group i n Several High-Spin I r o n ( I I I ) Porphyrins Distance (A) D e r i v a t i v e (Fe..N) av Ct...N Ct...Fe Fe-X Ref ClFeProtoDME 3 2.062(10) 2,008 0.475. 2.218(6) 41 CH3OFeMesoPDMEb 2.073(6) 2.022 0.455 1.842(4) 23 ClFeTPP 2.049(9) 2.012 0.38 2.192(12) 40 0(FeTPP) 2 2.087(5) 2.027 0.50 1.763(1) 48 (SCN)FeTPP 2.065(5) 2.007 0.485 1.957(5) 49 N^FeTPP 2.055 2.027 0.34 1.909 50 0(FeProtoDME) 2 a 2.08 - - 1.73 51 0(FeODM) 2 c 2.065(8) 2.002 0.53 1.752(1) 52 ^ Dimethyl ester of protoporphyrin Dimethyl e s t e r of mesoporphyrin a,Y-Dimethyloctaethylporphyrin From Scheidt (Ref. 47). -21-The stereochemical p a t t e r n i s maintained f o r an F e ( I I I ) porphyrin d e r i v a t i v e i n which two non-equivalent a x i a l l i g a n d s are coordinated. o In N 3(Py)FeTPP (46), the F e ( I I I ) i o n i s centered to w i t h i n 0.03A and has o a shortened (Fe-N) d i s t a n c e of 1.990A. av The low-spin F e ( I I ) porphyrins e x h i b i t stereochemical behavior s i m i l a r to F e ( I I I ) d e r i v a t i v e s . The F e ( I I ) i o n i n the centrosymmetric Pip„FeTPP (31) i s e x a c t l y centered. The e q u a t o r i a l (Fe-N) bond ^ av o d i s t a n c e i s 2.004A which i s s l i g h t l y l a r g e r than the c h a r a c t e r i s t i c low-s p i n F e ( I I I ) d e r i v a t i v e s . 2.1b Cobalt Porphyrins A l l c o b a l t porphyrins, e i t h e r c o b a l t ( I I I ) , d^, or c o b a l t ( I I ) , d^, are low-spin complexes. U n l i k e i r o n porphyrins, c o b a l t complexes do not e x h i b i t s p i n - s t a t e t r a n s i t i o n s . The stereochemistry of c o b a l t porphyrins i s g r e a t l y i n f l u e n c e d by the o x i d a t i o n s t a t e s and the number of a x i a l l i g a n d s . Table 2.5 gives the stereochemical parameters of f i v e - c o o r d i n a t e low-spin c o b a l t ( I I ) porphyrin d e r i v a t i v e s . Table 2.3 shows that d i s p l a c e -ment of the metal atom from the mean porphinato n i t r o g e n plane occurs only o i f the complexing M-N > 2.01A. An i n s p e c t i o n of Table 2.5,. however, i n -d i c a t e s that c o b a l t porphyrins do not obey the above observation; the cobalt atom i s d i s p l a c e d from the mean.plane of the p y r r o l e nitrogens by the same dista n c e s i r r e s p e c t i v e whether the M-N bond, d i s t a n c e i s greater o or l e s s than 2.01A. With an unoccupied d^2_y2 o r b i t a l , the c o b a l t ( I I ) i s a p p r o p r i a t e l y found to have a smaller out-of-plane displacement than h i g h - s p i n i r o n porphyrins. -22-TABLE 2.5 Stereochemical Parameters of the Square-Pyramidal Coordination Group f o r Low-Spin C o b a l t ( I I ) Porphyrins o Distance (A)  D e r i v a t i v e (Co-N) Co-N a Co...P„ Co...P N ...P Ref. av ax N c ax c NMeimCoTPP 1, .977(3) 2. •157(3) 0.. .13 0.14 2. .30 54 NMelmCoOEP 1. .96(1) 2. .15(1) 0. .13 0.16 2, .33 55 (3,5-Lut)CoTPP 2, .000(5) 2. ,161(5) 0. .14 0.17 2. .33 56 DiMeImCoTPPb 1, .985(2) 2. ,216(2) 0. .15 0.18 2. .37 57 N i s the n i t r o g e n atom of the a x i a l l i g a n d ax DiMelm i s 1,2-dimethylimidazole From Scheidt (Ref. 47) The P i p 2 C o ( I I I ) T P P + (Co,d 4) c a t i o n (53) and low-spin P i p 2 F e ( I I ) T P P 4 (Fe,d ) are both i s o e l e c t r o n i c and i s o s t r u c t u r a l . Both porphinato cores o are e s s e n t i a l l y planar. The ( C o - N ) ^ bond d i s t a n c e i s 1.978A compared to o the(Fe-N) of 2.004A. The d i f f e r e n c e between the two M-N dista n c e s i s av the d i r e c t consequence of the d i f f e r i n g nuclear charge on the two i o n s . The porphinato core of 0 2N(3,5-Lut)CoTPP (36) i s extremely r u f f l e d o w i t h the corresponding short ( C o -N) a v d i s t a n c e of 1.954A which probably represents the minimum Co(III)-N d i s t a n c e . The a x i a l Co-N^.^ di s t a n c e i s o 1.948A as opposed to the a x i a l Co-N,., c _ . di s t a n c e of "2.036A. The d i f f e r e n c e (3,5-Lut) -23-i n the two bond lengths p r i m a r i l y r e f l e c t s the d i f f e r i n g s t e r i c requirements of the two a x i a l l i g a n d s . - 2 4 -2.2 Application of Metalloporphyrin Stereochemistry to Heme  Stereochemistry i n Hemoproteins (Myoglobin and Hemoglobin) This section concerns the application of metalloporphyrin stereo-chemistry discussed i n Section 2.1 to heme stereochemistry i n myoglobin and hemoglobin. The objective i s to show how structural determination of metalloporphyrins can provide an insight into the mechanism of reversible oxygenation i n hemoglobin and myoglobin. I t must be pointed out that there i s s t i l l a l o t of controversy and continuing debate concerning the a l l o s t e r i c model proposed by Perutz on the oxygen-binding co-operativity phenomenon i n hemoglobin (28,58). In hemoglobin the ferrous heme i s d i r e c t l y anchored to the globin (protein) framework through the a x i a l complexing bond formed by the iron(II) atom with an imidazole nitrogen atom of the proximal h i s t i d i n e residue (F8). In the low-spin, oxy-Hb, molecular oxygen occupies the vacant si x t h position i n the coordination group of the iron atom. There i s no si x t h ligand i n the high-spin, deoxy-Hb. When the structure of hemoglobin was f i n a l l y solved, the hemes (59-63) were found to l i e i n isolated pockets with entrances on the surface of the subunits. The minimum spacing between any two heme iron o atoms i n deoxy-Hb or oxy-Hb i s >25A (28,6). Direct heme-heme interactions hence are an implausible source of co-operative phenomenon. As Perutz (6) posed the question "without contact between them (hemes) how could one of them sense whether the others had combined with oxygen?". Yet communication between the hemes i n each subunit i s prerequisite because a l l the co-operative effects disappear i f the hemoglobin molecule i s merely s p l i t i n half. I t was noted by Hoard i n 1966 (27) that the probable s t a r t i n g point f o r a stereochemical mechanism of co-operative i n t e r a c t i o n was the move-o ment >0.5A of the i r o n atom r e l a t i v e to the porphinato core upon oxygenation accompanied by the concomitant h i g h - s p i n to low-spin t r a n s i t i o n . In Perutz's subsequently developed mechanism (28,29) f o r the co-operative oxygenation of o hemoglobin, the primary t r i g g e r i s the shrinkage of about 0.90A i n the Py...Ne di s t a n c e that accompanies the t r a n s i t i o n from h i g h - s p i n f i v e -c o o r d i n a t i o n i n deoxy-Hb to low-spin s i x - c o o r d i n a t i o n i n oxy-Hb. I t i s important to note that only the mean plane, Py, of the e n t i r e macrocycle i n deoxy-Hb that can be i d e n t i f i e d ; the protoporphyrin i s viewed "edge-on as a r a t h e r t h i c k band of unresolved e l e c t r o n d e n s i t y i n which a s u b s t a n t i a l doming of the porphyrin core toward, t h e . i r o n atom may be wholly obscured". M-N distance i n both (non-equivalent) a- and 3-subunits of deoxy-Hb are o estimated to be ^0.6A. According to Perutz's proposal, there must be a " t e n s i o n " i n the a x i a l connection between the ferroheme and the g l o b i n ( p r o t e i n ) . A comparison of the Py...Ne di s t a n c e i n deoxy-Hb w i t h an e x t e r n a l l y unconstrained, f i v e - c o o r d i n a t e . 2-MeImFe(II)TPP (64,65) provides d i r e c t evidence f o r Perutz's proposal. I t i s i n t e r e s t i n g to note that s t e r i c a l l y hindered 2-Methyl imidazole r a t h e r than u n s u b s t i t u t e d imidazole i s used as -the a x i a l ligand i n the high-spin five-coordinate species. The reason i s to preclude the formation of the low-spin s i x - c o o r d i n a t e species. Py...P£ i s the perpendicular d i s t a n c e separating the a x i a l n i t r o g e n atom (Ne) of the heme-linked h i s t i d i n e (F8) from the mean plane (Py) of the ferroheme. - 2 6 -Th e Pu...Ne di s t a n c e i s 2.90A i n deoxy-Hb as opposed to 2.60A i n the unconstrained h i g h - s p i n , f i v e - c o o r d i n a t e 2-MeImFe(II)TPP (64). o This corresponds to a shortening of the Pu...Ne by 0.30A i n 2-MeImFe(II)TPP r e l a t i v e to the Pu...Ne i n deoxy-Hb. In other words, the a x i a l connection between the ferroheme and the g l o b i n i s i n te n s i o n . Another piece of evidence comes from the h i g h - s p i n (l-Melm)Mn(TPP) complex (66). The o o Mn...Pc di s t a n c e i s 0.515A and the doming parameter i s s t i l l only 0.04A o (66) as compared to Fe...Pu=0.75A i n horse deoxy-Hb. Unless the Fe...Pu o d i s t a n c e i n deoxy-Hb i s overestimated by at l e a s t 0.25A, i t appears that the a x i a l connection must be i n te n s i o n . H o p f i e l d (67) has quoted an o e r r o r of ±0.1A i n connection w i t h the out-of-plane displacement of the Fe ( I I ) atoms i n deoxy-Hb. Some f u r t h e r evidence i n support of the te n s i o n model come from n.m.r. stu d i e s of normal and abnormal - hemoglobin's (68-73). L i t t l e and Ibers (55) have discussed the pros and cons of the n.m.r. and resonance Raman s t u d i e s . o Perutz (6,62) estimated that the Fe atom i s d i s p l a c e d by 0.75A from the mean plane of the 24-atoms porphyrin s k e l e t o n . i n horse deoxy-Hb. I t o has been estimated that as much as ^0.20A i s due to doming of the core o toward the Fe atom l e a v i n g 0.55A to the out-ofT-plane displacement of the The Pu...Ne i n deoxy-Hb i s the summation.of Fe...Pu=0.75A and Fe...Ne=^2.15A. The Pu...Ne i n 2-MeImFe(II)TPP i s obtained by the summation of Fe...P =0 . 4 2 1 , P N >..P c=0.05A and Fe...Ne=2.16(Cos 10.3)A where 10.3 i s the angle o f t i l t of t h i s bond from the normal. -27-o i r o n (64). The magnitude of the doming of 0.20A can be c a l c u l a t e d as f o l l o w s : The stereochemical parameters f o r 2T-MeImFe(II)TPP are: Fe-Np=2.086A, o Fe...P N=0.42, and Ct...Np=2.044A. I f however, the Ct...Np. d i s t a n c e i s o decreased to 2.G1A (minimum e q u a t o r i a l s t r a i n to the macrocyle r i n g ; i n o human deoxy-Hb, i t i s 2.0-A (74)) w h i l e maintaining the Fe-Np d i s t a n c e at o 2.086A, from the Pythagorean theorem, the Fe...P displacement can be o o c a l c u l a t e d to be 0.55A. In other words,> Q.20A i s to be assigned to doming of the core. As can be seen from Figure 2.6, t r a n s i t i o n from deoxy-Hb to oxy-Hb o i n v o l v e s a shrinkage of ^G.9A i n the Eu...Ne which i s the sum of three -28-components: (a) a decrease i n Fe-Ne a x i a l bond, (b) a movement of the i r o n atom toward the Py plane, and (c) the movement re q u i r e d to b r i n g Py and P^ planes i n t o coplanar or near coplanar. I t i s important to point out the c a l c u l a t i o n i n v o l v e s bond dist a n c e s and displacements of the metal atoms taken from appropriate metalloporphyrins (55), The s t r u c t u r e of oxy-Hb i s s t i l l a w a i t i n g ; F e ( I I ) a u t o x i d i s e s r e a d i l y to F e ( I I I ) g i v i n g met-Hb. The replacement of the n a t u r a l p r o s t h e t i c group i . e . i r o n proto-porphyrin IX w i t h d i f f e r e n t metalloporphyrins i n myoglobin and hemoglobin has proved to be a u s e f u l approach (75). . Metalloporphyrins c o n t a i n i n g z i n c (75,76), manganese (77-81), copper (77), and n i c k e l (82) have been r e c o n s t i t u t e d i n t o hemoglobins. Their p r o p e r t i e s have been stud i e d and compared w i t h the n a t i v e hemoglobin. These r e c o n s t i t u t e d p r o t e i n s are incapable of r e v e r s i b l e oxygenation. Hoffman and a s s o c i a t e s (83-86) have shown that c o b a l t s u b s t i t u t e d myoglobin . (CoMb) and hemoglobin (CoHb) can combine r e v e r s i b l y and c o o p e r a t i v e l y w i t h molecular oxygen. The oxygen a f f i n i t y i s reduced by 10-100 times f o r CoMb and.CoHb compared to n a t i v e Mb and Hb (86). Since low-spin Co(II) e x h i b i t s a s m a l l e r . i o n i c r a d i u s than does high-spin. F e ( I I ) , Hoffman (83-85) p r e d i c t e d the displacement, of the Co(II) atom i n CoHb would be r e l a t i v e l y s m a ll compared w i t h that of the F e ( I I ) i n deoxy-Hb. The s t r u c t u r e of the low-spin, f i v e - c o o r d i n a t e 1-MeImCo(II)TPP was subsequently determined by Scheldt (54), As p r e d i c t e d by Hoffman, o Scheldt found that the cobalt atom.is d i s p l a c e d 0.13A from the mean plane o of the porphinato nitrogens and the doming (Py.-.P^) i s only ^O.OIA; o consequently, the c o b a l t atom i s d i s p l a c e d by ^0.14A from the mean plane •N he d e o x y - H b T 2 . 2 7 \ \ 2 - 8 2 \ 0 . 5 5 A=0.85A •N- he 1 . 9 7 A N-V N Ne C o 2I15 | } 2 . 3 1 A d e o x y - C o H b A = 0 . 3 8 A 1.93A • N — C o — N -0 F i g u r e 2 . 6 . I l l u s t r a t i o n o t c a l c u l a t i o n ot t h e t o t a l m o v e m e n t o f t h e p r o x i m a l h i s t i d i n e i n H b ( t o p ) a n d C o H b ( b o t t o m ) . T h e d i s p l a c e m e n t s a r e t a k e n r e l a t i v e t o t h e 2 4 - a t o m p o r p h y r i n c o r e . F r o m L i t t l e & I b e r s ( R e f . 5 5 ) i NJ VO I -30-of the porphyrin. S i m i l a r l y , i n the low-spin, f i v e - c o o r d i n a t e 1-MeImCo(II)OEP, o L i t t l e and Ibers (55) found the Co atom i s d i s p l a c e d by 0.13A from the mean o plane of the p y r r o l e n i t r o g e n atoms and 0.16A from the mean plane of the porphyrin core. I t appears that the out-of-plane displacement of the Co o atom i n CoHb i s u n l i k e l y to exceed 0.2A. On t h i s b a s i s , Hoffman et a l . , (83), and L i t t l e et a l . , (55) questioned whether the r e l a t i v e l y s m a ll out-of-plane displacement of t h e Co atom would be c o n s i s t e n t w i t h t h e t r i g g e r mechanism f o r cooperative oxygenation proposed by Pertuz (28,29). The unpaired e l e c t r o n i n the 3d^2_y2 °tbital of the h i g h - s p i n o F e ( I I ) atom accounts f o r the s u b s t a n t i a l Fe..,P displacement of 0.42A i n 2-MeImFe(II)TPP, whereas the absence of such an e l e c t r o n i n the low-o s p i n Co(II) atom allows the Co...P^ displacement to be 0.13A i n the l-MeImCo(II)TPP (54) and 1-MeImCo(II)OEP (55). In other words, i n deoxy-CoHb, the Co atom i s already c l o s e r to the mean porphyrin plane than Fe atom i n deoxy-Hb and i s l i k e l y to have created a r e a l s t r a i n i n the Co-Ne connection. This i s demonstrated by the lengthening of o the Co-Ne bond i n DiMelmCoTPP (Co...P =0.15A) r e l a t i v e to 1-MeImCoTPP N o (Co..,P^=0.13A) as shown i n Table 2.5. More s t r e t c h and enhanced t e n s i o n i n the a x i a l connection w i l l r e q u i r e greater s t r a i n i n the g l o b i n i n deoxy-CoHb than i n deoxy-Hb and consequently a r e d u c t i o n i n the Pu...Ne sepa r a t i o n r e a l t i v e to that i n deoxy-Hb. The above argument assumes that the g l o b i n can support increased t e n s i o n i n Co-Ne bond so that quaternary s t r u c t u r e of deoxy-Hb i s r e t a i n e d . Figure 2.6 a l s o i l l u s t r a t e s the c a l c u l a t i o n of the t o t a l movement of the proximal h i s t i d i n e i n Hb and CoHb by L i t t l e and Ibers (55). However, i t i s important to point out that no doming of the co b a l t porphinato core i s assumed. The t o t a l movement of the proximal h i s t i d i n e -31-i s estimated to be 0.38A which i s approximately h a l f that f o r Hb. On t h i s b a s i s , L i t t l e and Ibers conclude that " i t i s u n l i k e l y that such a o small movement (0.38A) could t r i g g e r the conformation changes observed i n Co-Hb". A counterproposal has been advanced by Hoard and Scheidt (64) that moderate doming of the porphinato core i n combination w i t h modest s t r e t c h i n the Co-Ne bond can r e a d i l y lead to an Py...Ne se p a r a t i o n o approaching 2.90A i n deoxy-Hb (Figure 2.7). In 1974, Hoard (65) proposed o o the doming to be 0.3A ins t e a d of 0.5A. The essence of the Hoard and Scheidt argument i s that t e n s i o n w i t h i n the p r o t e i n i n deoxy-CoHb not only causes the porphyrin to undergo s u b s t a n t i a l doming but a l s o increases the Co-Ne(histidine) bond. The doming r a i s e s the Co atom out-of-plane o o displacement to ^ 0.70A compatible to the Fe atom (0.75A) i s horse deoxy-Hb. Results from s e v e r a l s t u d i e s (87,88) are against the t e n s i o n model i n Co-Hb proposed by Hoard et a l . , (64). Resonance Raman stu d i e s of CoHb and Hb by Woodruff et a l . , (87) i n d i c a t e that the Co(II) atom out-o of-plane displacement i n deoxy-CoHb cannot exceed 0.2A. Besides, the resonance Raman spectrum of deoxy-CoHb i s e s s e n t i a l l y the same as f o r l-MeImCo(II)OEP. EPR experiments w i t h CoHb and model systems (88) a l s o gives no convincing i n d i c a t i o n s of te n s i o n w i t h i n the g l o b i n that would increase the out-of-plane displacement of the Co atom or s t r e t c h the Co-Ne ( h i s t i d i n e ) bond i n CoHb. However, the s t r u c t u r e of spermwhale oxymyoglobin r e c e n t l y determined by P h i l l i p s (26) provides f u r t h e r evidence f o r Perutz's a l l o s t e r i c model. The X-ray data was c o l l e c t e d at -12°C to prevent a u t o x i d a t i o n of F e ( I I ) to F e ( I I I ) g i v i n g met-Mb. P h i l l i p s found that the i r o n atom i s d i s p l a c e d N. •N N: 2.15 A 0.13 0.03 C o ( 1 - M e - I m ) ( O E P ) 2.25 A C o H b F i g u r e 2 . 7 . I l l u s t r a t i o n o f p o s s i b l e p o i n t s o f d i s t o r t i o n o f t h e C o ( I I ) p o r p h y r i n p r o s t h e t i c g r o u p a s p r o p o s e d b y H o a r d & S c h e i d t ( 6 A ) . F r o m L i t t l e & I b e r s ( R e f . 5 5 ) i UJ ho I -33-from the mean porphyrin plane by 0.26A towards the proximal h i s t i d i n e (F8), and the bound oxygen has the bent-geometry. So f a r there i s no measure of the corresponding Fe displacement i n oxy-Hb because under the normal c o n d i t i o n s of X-ray c r y s t a l l o g r a p h y , oxy-Hb i s r e a d i l y o x i d i s e d to met-Hb. I t i s a common n o t i o n that the Fe atom i s coplanar w i t h the heme plane i n oxy-Hb, however, the s t r u c t u r e of oxy-Mb i n d i c a t e s t h i s i s not the case. An i n t e r e s t i n g f e a t u r e of oxy-Mb i s that the heme-linked imidazole of h i s t i d i n e (F8) and Fe-0-0 are approximately coplanar and e l c i p s e d w i t h the Fe-N bond of the p y r r o l e I I r i n g . I t seems probable the s t e r i c arrangement of the h i s t i d i n e (F8) and Fe-0-0 plane that prevent the Fe atom from being coplanar w i t h the porphyrin plane. Table 2.6 shows that the t r a n s i t i o n from deoxy-Mb to oxy-Mb i n v o l v e s o the movement of the Fe atom towards the heme plane and a shrinkage of 0.59A o i n the Pu...Ne as opposed to ^ 0.90A i n the t r a n s i t i o n from deoxy-Hb to met-Hb. The d i f f e r e n c e i n shortening of the Pu...Ne i s conceivably a t t r i -buted to the f a c t that myoglobin i s a s i n g l e polypeptide which hence does not e x h i b i t oxygen-binding c o - o p e r a t i v i t y . P h i l l i p s (26) c l e a r l y demon-s t r a t e s that the Fe atom does move towards the porphyrin on oxygenation as expected according to Perutz's a l l o s t e r i c model (6,62). TABLE 2.6 Heme geometry i n Mb and Hb d e r i v a t i v e s Deoxy-Mb Oxy-Mb Met-Mb CO-Mb Deoxy-Hb Met-Hb Fe-(heme plane) 0.55 0.26* 0.40 0.24 0.60(a-heme) 0.07(a-heme) 0.63(8-heme) 0.21(g-heme) Fe(Ne)-(heme-plane) 2.6 2.3 2.5 2.4 2.6(a-heme) 2.2(a-heme) 2.8(g-heme) 2.4(g-heme) Values are given i n A *Value from r e f i n e d model ( P h i l l i p s , p r i v a t e communication) Adapted from P h i l l i p s (Ref.26) -34-2.3 Myoglobin and Hemoglobin - The Heme Group/Globin S t r u c t u r e The d e t a i l s of the s t r u c t u r e of myoglobin are mostly derived from the impressive X-ray c r y s t a l l o g r a p h i c s t u d i e s of J . C. Kendrew et a l . , (44, 89,90), and the s t r u c t u r e of hemoglobin from M. F. Perutz et a l . , (61,62). Myoglobin of 17,000 molecular weight represents the simplest hemo-p r o t e i n capable of r e v e r s i b l e oxygenation. This p r o t e i n contains only one polypeptide chain of 153 residues and one heme group per molecule. In c o n t r a s t , mammalian hemoglobins have a molecular weight of near 65,000 and are made up of four polypeptide chains, i d e n t i c a l i n p a i r s (a and B c h a i n s ) . The a-chains have 141 amino a c i d residues each w h i l e the $-chains have 146 residues each. Each chain harbors one heme ( f e r r o - p r o t o -porphyrin IX, .Figure 2.8) which when oxygenated, gives blood i t s red c o l o r . A s i n g l e polypeptide chain combined w i t h a s i n g l e heme i s c a l l e d a subunit of hemoglobin. I t i s evident, however, that the hemo-g l o b i n subunits and myoglobin have a s i m i l a r o v e r a l l s t r u c t u r e , and t h a t , more s p e c i f i c a l l y , the b a s i c l i n k a g e s between the heme and p r o t e i n are e s s e n t i a l l y the same (92-94). I t i s hence f e l t that hemoglobin i s a p p r o p r i a t e l y included i n t h i s s e c t i o n . The p r o s t h e t i c group of myoglobin and hemoglobins i s a protoheme (Figure 2.8) i n which the i r o n i s s t r o n g l y coordinated to the fou r p y r r o l e nitrogens of the protoporphyrin IX. Ferro-protoporphyrin IX i s known as heme; F e r r i - p r o t o p o r p h y r i n IX~ as hemin'. The f i f t h s i t e of the heme i s l i n k e d to the imidazole n i t r o g e n of a h i s t i d i n e r e s i d u e , number 93 of the myoglobin chain, number 87 of the hemoglobin a-chain, and number 92 of the hemoglobin 3-chain (44,89,90). The s i x t h , and l a s t , of the i r o n c o o r d i n a t i o n s i t e s i n heme i s a v a i l a b l e f o r oxygen or l i g a n d b i n d i n g (Figure 2.9). D i r e c t l y -35-CH 2=CH | CHj CHj C H 2 - C H 2 - C 0 0 H 6 I X N 3 Fe — N , 5 F i g u r e 2.8. F e r r o - p r o t o p o r p h y r i n IX (heme) and i t s c o o r d i n a t e s y s t e m . H i H 0 I l l 1 — N — l II c -C -| H — C -H i 1 C = C — 1 1 H — 1 1 N N s \ // C H E M E P L A N E F i g u r e 2 . 9 . A t t a c h m e n t o f heme i r o n t o N « o f h i s t i d i n e F 8 . L r e p r e s e n t s a l i g a n d i n t h e s i x t h p o s i t i o n . -36-opposite to the iron-linked imidazole is the "distal histidine", number 64 in myoglobin, number 58 in the hemoglobin a-chain, and number 63 in the B-chain of hemoglobin. It has been suggested that the dis t a l histidine may play an important role in the reversible oxygenation process. Recent crystallographic refinement of spermwhale metmyoglobin to o o 2.OA shows that the iron atom is displaced by 0.40A from the mean plane o of the heme, Py, and the Fe-Ne (proximal histidine) i s 2.13A (95). The o doming of the porphinato core is 0.13A which is in agreement with that found in 2-MeImFe(II)TPP (64). Takano (95) reported that the displacement o of the iron atom from the mean plane of the heme increases from 0.40A to o 0.55A on going from met-to deoxy. The structure of oxy-Mb was recently solved.by Phi l l i p s in 1978 (26). The Fe atom out-of-plane displacement from the mean plane of o the porphyrin i s 0.26A towards the proximal histidine (F8). Table 2.6 gives the heme geometry in Mb and Hb derivatives. Recently G. Fermi (74) obtained a refined structure of human deoxy-o Hb at 2.5A resolution using the electron-density map obtained by Ten Eyck and Arnone (96). The displacement of the iron atom from Py is less than that previously reported for horse deoxy-Hb (62), but the discrepancy 0 probably l i e s within the measurement errors. The Fe...Py is 0.60A in the o o a-heme and 0.63A in the B-heme. The estimated error is ±0.1A. With the o available 2.5A data, Fermi could.not assess the doming magnitude accurately. Fermi, however, pointed out that the doming of the porphinate core is o unlikely to exceed the value of 0.17 to 0.22A. The Py...Ne (proximal o o histidine) is 2.6A which is compatible with the value of 2.68A quoted by Hoard and Scheidt (64) for high-spin ferrous five-corrdinate 2-MeImFe(II)TPP. -37-In horse met-Hb, Bolton and Perutz (62) found the i r o n atom to be displaced from the mean plane of the porphyrin s k e l e t a l towards the o heme-linked h i s t i d i n e by 0.3A. In both the a- and g-chains of horse o deoxy-Hb t h i s displacement i s increased to 0.75A. This observation formed part of the experimental basis for Perutz's t r i g g e r mechanism of oxygen-binding co-operative phenomenon (6,29). The o v e r a l l shape of myoglobin i s a flattened sphere. The heme group i s near the surface of the molecule i n a non-polar, hydrophobic pocket formed by the amino acids, Ala, I l u , Phe., Leu, and Val. Figure 2.10 gives the stereo drawing of myoglobin. The presence of several nearly aromatic rings such as Phe p a r a l l e l to the heme, introduces the p o s s i b i l i t y of ir-bonding i n t e r a c t i o n s . The plane of the heme group i s approximately normal to the surface of the myoglobin. The heme i s i n van der Waals contact with 83 atoms, of the globin (excluding hydrogens). The hydrophilic chains are directed towards the surface to in t e r a c t with the solvent. The structure of the native protein appears to be held together e s p e c i a l l y by non-polar in t e r a c t i o n s between sidechains, and between sidechains and the heme group, with polar i n t e r a c t i o n s contributing to a lesser extent. About 70 to 80 per cent of the polypeptide chain i s arranged as an a-helix. In the a - h e l i c a l regions, the non-polar residues repeat at very regular i n t e r v a l s , every 3.6 residues, so that the side of the h e l i x which faces the i n t e r i o r of the molecule i s composed of a row of non-polar residues. The X-ray studies have revealed that each of the four subunits i n hemoglobin has a structure s i m i l a r to that of myoglobin, e s p e c i a l l y i n regard to the f o l d i n g of the polypeptide chain, the number and d i s t r i b u t i o n -38-F i g u r e 2.10. A t h r e e - d i m e n s i o n a l s t e r e o - p a i r d r a w i n g m y o g l o b i n . From D i c k e r s o n and G e i s ( R e f .97) • -39-of the h e l i c a l and n o n - h e l i c a l segments, and the p o s i t i o n and o r i e n t a t i o n of the heme group. The four hemoglobin subunits are arranged to form a n e a r l y r e g u l a r tetrahedron to give the whole molecule a s p h e r i c a l appearance (Figure 2.11). Again the heme groups are near the sur f a c e . The a- and 3-chains are complementary, and these chains are i n c l o s e contact, i n con t r a s t to the much lo o s e r a s s o c i a t i o n between the i d e n t i c a l chains. A major d i f f e r e n c e between myoglobin and hemoglobin i s that myoglobin e x h i b i t s no co-operative i n t e r a c t i o n w i t h molecular oxygen. -40-Figure 2.11. The folding and packing of chains in hemoglobin. From Dickerson and Geis (Ref.97). -41-2.4 Models f o r Myoglobin and Hemoglobin Several groups of i n v e s t i g a t o r s have developed models th a t mimic the a c t i v e - s i t e ( i . e . heme of myoglobin and hemoglobin. The s y n t h e t i c complexes are capable of r e v e r s i b l e oxygen-binding but i n non-aqueous media and o f t e n at very low temperatures, t y p i c a l l y -40°C. A good review a r t i c l e on s y n t h e t i c oxygen-carriers i s by B. R. James (98) i n the t r e a t i s e c o n s i s t i n g of seven volumes on porphyrins e d i t e d by D. Dolphin (99). Studies on such model systems would give in f o r m a t i o n about the nature of the metal-dioxygen bond and the ass o c i a t e d k i n e t i c and thermodynamic f a c t o r s . I s o l a t e d heme i s r a p i d l y o x i d i s e d to F e ( I I I ) w i t h the s i x t h s i t e of the i r o n c o o r d i n a t i o n s i t e s occupied by a hydroxyl i o n or c e r t a i n other l i g a n d s so that i t cannot bind oxygen.~ The i r r e v e r s i b l e o x i d a t i o n to F e ( I I I ) i s b e l i e v e d to proceed through an intermediate i n which two i r o n porphyrins are c o v a l e n t l y l i n k e d known as y - o x o - b i s ( p o r p h y r i n ( I I I ) ) as shown i n Figure 2.12. This creates a major ob s t a c l e i n designing a s u i t a b l e h i g h - s p i n f i v e - c o o r d i n a t e F e ( I I ) complex as a p r o t e i n - f r e e model f o r myoglobin. Several models have been synthesized i n the recent years (100-102). An important point to note i s that a l l the model compounds so f a r reported are monomers; hence, they are more c o r r e c t l y considered to be models of myoglobin r a t h e r hemoglobin. In a l l of the model systems, oxygen-binding can be accomplished only when the f i f t h s i t e of the c o o r d i n a t i o n of the metal atom i s c o v a l e n t l y bound to a base, g e n e r a l l y , an imidazole. This leaves only the s i x t h c o o r d i n a t i o n s i t e of the metal atom a v a i l a b l e f o r oxygen-binding. -42-F i g u r e 2.12 A s c h e m a t i c d r a w i n g o f y - o x o - b i s ( p o r p h y r i n i r o n ( I I I ) ) N N \ F / 0' / Fe Fe \ N N I n h e m o g l o b i n and m y o g l o b i n , t h e hemes a r e embedded i n n o n - p o l a r , h y d r o p h o b i c p o c k e t s as d i s c u s s e d i n S e c t i o n 2.3. X - r a y c r y s t a l l o g r a p h i c s t u d i e s (6,28) show th e d i s t a n c e between any two i r o n atoms i n deoxy-Hb o r oxy-Hb i s >25A; t h a t p r o b a b l y e x p l a i n s why t h e oxygen m o l e c u l e b i n d s t o F e ( I I ) atom i n h e m o p r o t e i n s w i t h o u t o x i d i z i n g t h e i r o n t o F e ( I I I ) . On t h i s b a s i s , C o l l m a n ' s group a t S t a n f o r d s y n t h e s i z e d a p o r p h y r i n r i n g w i t h f o u r b u l k y o - p i v a l a m i d e p h e n y l groups p r o j e c t i n g f r o m one s i d e o f t h e r i n g l i k e s t a k e s i n a p i c k e t f e n c e , hence, known as " p i c k e t f e n c e " p o r p h y r i n (100,103,104,105). F i g u r e 2.13 i l l u s t r a t e s t h e " p i c k e t f e n c e " p o r p h y r i n . The p u r p o s e o f t h e b u l k y s u b s t i t u e n t s i s t o p r e v e n t t h e i n t i m a t e c o n t a c t by t h e Fe atoms n e c e s s a r y f o r o x i d a t i o n . I n s t e a d o f a f i v e c o -o r d i n a t e s p e c i e s , a h e x a - c o o r d i n a t e F e ( I I ) complex w i t h two n i t r o g e n o u s b a s e s as t h e a x i a l l i g a n d s i s p r o d u c e d . I n benzene a t 20°C, t h e i m i d i a z o l e complexes undergo r e v e r s i b l e o x y g e n a t i o n ( 9 8 ) , a l t h o u g h i r r e v e r s i b l e o x i d a t i o n o c c u r s s l o w l y . o -43-T r a y l o r ' s group (101) and Baldwin's group (102) have a l s o constructed s e v e r a l l y s t e r i c a l l y hindered porphyrins designed to prevent d i m e r i z a t i o n . These complexes are capable of r e v e r s i b l e oxygenation. Baldwin and a s s o c i a t e s (102) reported the synthesis of "capped" porphyrins (Figure 2.14). The "cap" prevents the formation of hexa-coordinate s p e c i e s , and molecular oxygen enters the c a v i t y on the protected s i t e . The complexes are capable of r e v e r s i b l e oxygen-binding i n p y r i d i n e at 25°C. Chang and T r a y l o r (106) synthesized.an i n s i t u f errous complex such as shown i n Figure 2.15 i n which an imidazole i s c o v a l e n t l y bound to a porphyrin r i n g . I t i s i n t e r e s t i n g to note that the geometry of the complex i s s i m i l a r to that of myoglobin. R e v e r s i b l e oxygen-binding occurs only at -45°C i n dichloromethane. -44-F i g u r e 2.13. " P i c k e t - f e n c e " p o r p h y r i n Collman e t a l . , (100,103-105) F i g u r e 2.14. "Capped" p o r p h y r i n s Baldwin e t a l . , (102) —»-—«*»)„• n C H - - C CH, CH. \ —C ^ C - C N j C M j C O j t , F i g u r e 2.15. C h e l a t e d hemes h a v i n g v a r y i n g degrees o f s t e r i c h i n d r a n c e toward base c h e l a t i o n . T r a y l o r e t a t . , ( 1 0 6 ) . -45-CHAPTER I I I EXPERIMENTAL WORK 3.1 P r e p a r a t i o n of a,g,Y,6-Tetraphenylphinatoindium(III) c h l o r i d e M a t e r i a l s : Indium c h l o r i d e , I n C l ^ (anhydrous, u l t r a - p u r e ) was obtained from A l f a ; meso-tetraphenylporphine was obtained from Strem Chemicals, Inc., and reagent g l a c i a l a c e t i c a c i d from A l l i e d Chemical Canada, L t d . S i l i c a g e l (70-140 mesh) f o r column chromatography was obtained from Macherey, Nagel & Co., Germany. Procedure: The procedure used to synthesize the indium meso-tetraphenylporphyrin c h l o r i d e (InTPPrCl) was e s s e n t i a l l y that of B h a t t i et a l . , (107). In a o n e - l i t r e one-necked round-bottom f l a s k f i t t e d w i t h a r e f l u x condenser, indium c h l o r i d e (0.002M) and meso-tetraphenylporphine (0.001M) were heated to b o i l i n g i n g l a c i a l a c e t i c a c i d (500 ml) c o n t a i n i n g 2.2 gm of sodium acetate. The r e a c t i o n was protected from l i g h t by aluminium f o i l . R e f l u x i n g was continued f o r about 4 hr. Completion of the m e t a l a t i o n process was determined s p e c t r o p h o t o m e t r i c a l l y by the disappearance of the 515 nm peak c h a r a c t e r i s t i c of the pure porphine and the appearance of a peak i n the 560 nm region c h a r a c t e r i s t i c of the indium porphine. Upon c o o l i n g to room temperature, l u s t r o u s , purple c r y s t a l s of InTPPrCl were obtained. The product was c o l l e c t e d by vacuum f i l t r a t i o n , washed w i t h methanol and a i r - d r i e d . The r e a c t i o n took place according to equation 3.1 H 2(TPP) + I n C l 3 + 2Na(0Ac) InTPPrCl + 2H(0Ac) + 2NaCl (3.1) -46-The o v e r a l l stoichiometry i n Eq. 3.1, looks rather simple. In fact there must be at l e a s t f i v e separate processes taking place i n Eq. 3.1. They are as follows: (a) Protonation/Deprotonation E q u i l i b r i a ?+ -4-?H~'~ _9ii"'~ o_ H.(TPP) Z^h H„(TPP) > (TPP) (3.2) 4 7 £ t 2-Deprotonation of H 2(TPP) w i l l give a dianion,(TPP) , which w i l l r e a d i l y complex with a p o s i t i v e l y charged metal ion. When meso-tetraphenyl-porphine was f i r s t dissolved i n g l a c i a l a c e t i c acid, a greenish s o l u t i o n was obtained. The greenish color was a t t r i b u t e d to the formation of a 2+ d i a c i d species, H^(TPP) (See Appendix B). As the reaction progressed, a deep purple s o l u t i o n was obtained i n d i c a t i n g the formation of InTPP:Cl. From Eq. 3.2, i t i s clear that strong acids w i l l impede or reverse metal-l a t i o n by s h i f t i n g the equilibrium to the l e f t . A weak acid such as g l a c i a l a c e t i c acid was used. At f i r s t glance, Eq. 3.2 suggests that basic solvents should be the more appropriate solvents than acids because they w i l l drive the depro-tonation equilibrium to the r i g h t . However, the basic solvent molecules behave as Lewis bases which may more or le s s strongly bind as neutral donors to the metal ions. This w i l l s h i f t the e q u i l i b r i a i n Eq. 3.3 to 3+ the l e f t , thus impeding the formation of "naked" In metal ions. Sodium acetate was added to buffer the s o l u t i o n and to enhance deprotonation of the porphyrins by s h i f t i n g the equilibrium (Eq. 3.2) to the r i g h t . (b) Deconvolution of the metal ion from the metal c a r r i e r InCl- + 3Na(0Ac) s In(OAc)., + 3NaCl (3.3a) -47-In(OAc) 3 , k I r T r r + 3(0Ac) (3.3b) The formation of I n ( T P P ) + depends on the a v a i l a b i l i t y of "naked" 3+ 2-In ions that w i l l complex w i t h "bare" (TPP) . This i m p l i e s 3+ that indium acetate has to d i s s o c i a t e r e a d i l y to give I n i o n s . This may be a very d e c i s i v e step i n the m e t a l a t i o n r e a c t i o n . (c) Formation of the e q u a t o r i a l MN^ species I n 3 + + ( T P P ) 2 _ ^ = ^ I n ( T P P ) + (3.4) Complexation between a "bare" In~ r T~ i o n and the d i a n i o n (TPP)^ i s a s t r a i g h t f o r w a r d r e a c t i o n . (d) N e u t r a l i z a t i o n of the charge I n ( T P P ) + + C l " * InTPPrCl (3.5) To maintain e l e c t r o n e u t r a l i t y , an anion i s bound to the metal i o n . I t i s a s t o n i s h i n g that i n s p i t e of the l a r g e excess of acetate present, acetate complex was not formed. (e) Completion of the c o - o r d i n a t i o n sphere 3+ The In i s c o o r d i n a t e l y saturated, thus only f i v e - c o o r d i n a t e square-pyramidal InTPPrCl i s formed. In some metal complexes, s i x - , and e i g h t -coordinate species are p o s s i b l e . -48-3.2a P u r i f i c a t i o n of InTPP:Cl The p u r i f i c a t i o n procedure f o r InTPPrCl was d i f f e r e n t from that of B h a t t i et a l . ; (107). The crude complex (100 mg) was d i s s o l v e d i n minimal amount of chloroform and chromatographed on a 62 cm x 1.6 cm column of s i l i c a g e l packed i n toluene. The column was e l u t e d w i t h a 1:1 toluene/chloroform/chloroform mixture, the unreacted TPP was eluted f i r s t , f ollowed by InTPPrCl, A l l f r a c t i o n s were checked s p e c t r o s c o p i c a l l y and by t i c . The solvent was evaporated on a vacuum r o t a r y evaporator to recover InTPPrCl. 3.2b T h i n - l a y e r Chromatography Thi n - l a y e r chromatography was used to check the p u r i t y of the p u r i f i e d InTPPrCl. A n a l y t i c a l t h i n p l a t e s of s i l i c a g e l (Eastman) were spotted w i t h 5 y l samples. A mixture of toluene/chloroform (1:1) was used to develop the chromatogram. The chromatograms were run i n the dark. TLC showed the absence of the s t a r t i n g m a t e r i a l , meso-TPP i n the p u r i f i e d InTPPrCl. 3.2c V i s i b l e Absorption Spectroscopy The v i s i b l e a b s o r p t i o n s p e c t r a of InTPPrCl and meso-TPP i n chloroform were recorded on a Cary 14 spectrometer (Figure 3.1 and Figure 3.2). The meso-TPP shows four absorption bands (excluding the Soret band) i n the v i s i b l e r e g i o n , l a b e l l e d I-IV (Figure 3.2). The very intense Soret band 5 —6 (e>10 ) could only be determined at a c o n c e n t r a t i o n of VLO M. On formation of InTPPrCl, the four-banded spectrum c o l l a p s e d i n t o an e s s e n t i a l l y two-banded one (a and 8 bands) i n the v i s i b l e r e g i o n , whereas the Soret band remained. These bands are bathochromically s h i f t e d , the a-band o c c u r r i n g at 600 nm, the 8-band at 560 nm, and the Soret band at 419 nm; the i n t e n s i t y sequence i s Soret >>g>a. -49-4 2 6 J 1 L I I i j 3 0 6 3 8 4 4 6 0 474 5 4 9 6 2 4 7 0 0 W a v e l e n g t h n m F i g u r e 3.1. V i s i b l e a b s o r p t i o n s p e c t r u m o f I n T P P : C l i n c h l o r o f o r m . -50-i IV 515 310 384 460 471 547 639 700 Wavelength nm F i g u r e 3.2. V i s i b l e a b s o r p t i o n s p e c t r u m o f m e s o - T P P i n c h l o r o f o r m . -51-3.2d Nuclear Magnetic Resonance Spectroscopy The n.m.r. spectrum of InTPPrCl i n deuterochloroform was recorded 2 on a V a r i a n XL-100 FT-NMR spectrometer, w i t h D-Lock on CDC1 3. 3.3 R e c r y s t a l l i s a t i o n of InTPPrCl S p e c t r o q u a l i t y chloroform was obtained from MCB, and spectro-methanol was obtained from Eastman. R e c r y s t a l l i z a t i o n was c a r r i e d out by d i s s o l v i n g the met a l l o p o r p h y r i n i n a minimal volume of chloroform, and adding methanol dropwise to the b o i l i n g s o l u t i o n . With continued b o i l i n g , chloroform d i s t i l l e d away, and the methanol con c e n t r a t i o n increased; the process was allowed to continue u n t i l some small c r y s t a l s formed. Purple s i n g l e c r y s t a l s were obtained by l e t t i n g the mixture stand f o r s e v e r a l days at room temperature. -52-CHAPTER IV RESULTS AND DISCUSSION 4.1 Metal displacement above mean p y r r o l e n i t r o g e n plane The c r y s t a l s are shown to be monoclinic w i t h a space group of P2^/n. The numbering scheme used f o r the carbon and n i t r o g e n atoms i n the asymmetric u n i t of c r y s t a l l i n e InTPPrCl i s shown i n Figure 4.1. The contents of the u n i t c e l l are shown i n Figure 4.2. The stereo-drawing of InTPP:Cl i n Figure 4.3 c l e a r l y shows the displacement of the indium atom above the mean plane of the four p y r r o l e n i t r o g e n atoms, and a l s o s i g n i f i c a n t doming of the porphinato core. The o indium atom out-of-plane displacement i s 0.71A from the mean plane of the o porphyrin w i t h 0.1A a t t r i b u t e d to the "doming". InTPPrCl i s not planar but e x h i b i t s a " r u f f l i n g " of the porphyrin skeleton. A number of i n t e r e s t i n g features emerge from the stereochemical data of v a r i o u s metalloporphyrins l i s t e d i n order of decreasing metal displacements (Table 4.7)r (a) there i s so simple c o r r e l a t i o n between the i o n i c radius and metal i o n displacement; (b) the displacements f o r the same metal coordinated to d i f f e r e n t porphyrins are s i m i l a r as evident from Co-o atom displacement of 0.13A both i n l-MeImCo(II)TPP (54) and 1-MeImCo(II)OEP (55), and the various h i g h - s p i n f e r r i c porphyrin complexes a l s o have s i m i l a r Fe...Py d i s t a n c e s ; and (c) i n d i u m ( I I I ) atom i s d i s p l a c e d e s s e n t i a l l y the same d i s t a n c e as i n h i g h - s p i n F e ( I I ) i n deoxy-Hb (horse or human). I t i s o f t e n i n f e r r e d that h i g h - s p i n s t a t e s imply f i v e - c o o r d i n a t i o n , w h i c h so f a r i s v a l i d f o r f e r r o u s porphyrins and hemoproteins. In 1978, Mashiko et a l . , ( I l l ) reported the f i r s t s t r u c t u r a l c h a r a c t e r i s a t i o n of s i x - c o o r d i n a t e h i g h - s p i n f e r r i c porphyrin d e r i v a t i v e s of TPP i n which the F e ( I I I ) atom i s F i g u r e 4 . 1 . A t o m i c n u m b e r i n g s y s t e m f o r c r y s t a l l i n e I n T P P r C l . The a t o m s a r e d r a w n as 50% p r o b a b i l i t y t h e r m a l e l l i p s o i d s . The H a t o m s h a v e b e e n o m i t t e d f o r c l a r i t y . The p h e n y l r i n g s a r e numbered a s n C m > w h e r e n i s t h e g r o u p number (n=l , 4 ) and m i s t h e r i n g c a r b o n ( m = l , 6 ) . F i g u r e 4 . 2 . S t e r e o s c o p i c v i e w o f t h e c o n t e n t s o f one u n i t c e l l o f c r y s t a l l i n e I n T P P : C 1 . F i g u r e 4.3. S t e r e o d i a g r a m o f I n T P P r C l , i l l u s t r a t i n g t h e In d i s p l a c e m e n t f r o m t h e m a c r o c y c l i c p l a n e , a n d t h e " d o m i n g " o f t h e p o r p h y r i n s k e l e t o n . The H a t o m s have been a r b i t r a r i l y r e d u c e d f o r c l a r i t y . -56-p r e c i s e l y i n the mean porphyrin plane. S i x - c o o r d i n a t i o n i s a l s o known i n h i g h - s p i n f e r r i c hemoproteins as i l l u s t r a t e d by aquo- and f l u o r o -methemoglobin. In c o n t r a s t , the F e ( I I I ) atoms are s i x - c o o r d i n a t e and low-spin i n CN-MetHb and CO-MetHb. The observation (b) emphasises the f a c t that the metal displacement behavior i s s i m i l a r i n a l l m e t a l l o p o r p h y r i n complexes of a s i n g l e metal type. I t i s l o g i c a l to assume w i t h confidence that subsequent f i v e - c o o r d i n a t e indium porphyrin complexes w i l l e x h i b i t s i m i l a r displacement of indium atoms above the mean porphyrin plane. Perutz (28,29) proposed a stereochemical mechanism to e x p l a i n the co-operative nature of oxygen-binding. The a l l o s t e r i c model was based on the d i f f e r e n c e s i n the t e r t i a r y and quaternary s t r u c t u r e s of horse deoxy-and met-Hb, r a t h e r than deoxy- and oxy-Hb. Oxy-Hb i s r e a d i l y o x i d i s e d to met-Hb under the normal c o n d i t i o n s of X-ray c r y s t a l l o g r a p h y . Perutz j u s t i f i e d h i s argument on the ground that oxy-Hb and met-Hb are isomorphous i . e . the c r y s t a l s have the same space group. Huestic and Raftery (112) 19 showed that the F - l a b e l l e d c y s t e i n e 938 i n met-Hb gives a n.m.r. chemical s h i f t s i m i l a r to deoxy-Hb but was d i f f e r e n t from oxy- and CO-Hb. This i m p l i e s that met-Hb i s not a good analog f o r oxy-Hb. Besides the F e ( I I I ) i s s i x - c o o r d i n a t e and predominantly high-spin i n met-Hb as opposed to F e ( I I ) which i s low-spin and s i x - c o o r d i n a t e i n oxy-Hb. Perutz i n 1976 (113) proposed that CO-Hb "may be regarded as the c l o s e s t r e l a t i v e of oxy-hemoglobin" on the ground that CO-Hb i s s t a b l e and isomorphous w i t h both oxy- and met-Hb. Furthermore, the F e ( I I ) atoms i n oxy- and CO-Hb o are low-spin. However, the 2.8A X-ray data on CO-Hb i s not s u f f i c i e n t to determine the exact displacement of the i r o n atoms from the heme plane. The s t r u c t u r e of spermwhale oxymyoglobin has been determined by P h i l l i p s i n -.57-1978 (26). P h i l l i p s c l e a r l y demonstrates that the Fe atom does move towards the porphyrin on t r a n s i t i o n from deoxy-to oxy-Mb (See Se c t i o n 2.2 and Table 2.6). This f i n d i n g r e i n f o r c e s Perutz's a l l o s t e r i c model although the t o t a l movement of the proximal h i s t i d i n e i n Mb i s smaller than that expected f o r Hb. Perutz (28,29) proposed that the quaternary s t r u c t u r e of hemoglobin i s i n e q u i l i b r i u m between two s t a b l e subunit c o n f i g u r a t i o n s termed as "T" f o r deoxy-Hb and "R" f o r oxy-Hb. In the deoxy-Hb, the low-spin, o f i v e - c o o r d i n a t e heme i r o n atom i s ^ 0.75A (horse hemoglobin) out of the heme plane towards the proximal h i s t i d i n e . When a molecule of oxygen binds to deoxy-Hb, the i r o n moves i n t o the plane of the porphyrin. According to Perutz's stereochemical mechanism, the t r a n s i t i o n from "T" s t a t e (deoxy-Hb) to "R" s t a t e (oxy-Hb) i n v o l v e s a shrinkage of M3.90A i n the Pu...Ne d i s t a n c e . This movement t r i g g e r s a s e r i e s of small changes i n the quaternary and t e r t i a r y s t r u c t u r e s of the p r o t e i n that r e s p o n s i b l e f o r the oxygen-binding c o - o p e r a t i v i t y . At the present there i s s t i l l a considerable debate and controversy surrounding the question of the stereochemical mechanism. There are two schools of thought: Perutz advocates that the changes i n the stereochemistry at the core of the heme are the key to the t r i g g e r mechanism w h i l e the other school ( E d e l s t e i n and Gibson (81(b)), and L i t t l e and Ibers (55))emphasises the importance of the i n t e r a c t i o n between the d i s t a l h i s t i d i n e and the s i x t h -l i g a n d . A c r i t i c a l t e s t f o r the t r i g g e r mechanism would be to " f r e e z e " one or more subunits i n t o the "T" s t a t e and then monitor changes i n the oxygenation k i n e t i c s , e l e c t r o n i c , and motional p r o p e r t i e s of the remaining i n t a c t n a t i v e subunit or subunits. Native subunits i n the "T" s t a t e are very -58-d i f f i c u l t to achieve s i n c e heme F e ( I I ) binds oxygen to become hexa-. coordinate and low-spin to give the "R" s t a t e . Since the "T" form cannot be s t a b l y generated using n a t i v e i r o n heme ( r e a d i l y a u t o x i d i s e s to F e ( I I I ) ) , other means must be sought. Various metalloporphyrins c o n t a i n i n g Mn, Co, Zn, and Mg i n place of na t i v e heme have been r e c o n s t i t u t e d i n t o hemoglobin. However, Mn(II) i s r e a d i l y o x i d i s e d to M n ( I I I ) , and Co(II) binds oxygen. Both Mg and Zn may be o x i d a t i o n s t a b l e but they e x h i b i t r e l a t i v e l y small metal displacements above the porphyrin plane r e l a t i v e to the Fe atom i n n a t i v e hemoglobin (See Table 4.7). The present X-ray data on InTPP:Cl suggest indium porphyrins are i d e a l candidates f o r inducing the "T" conformation ( v i v e i n f r a ) . I n c o r p o r a t i o n of indium porphyrins i n t o hemoglobin provide an opportunity to probe the prophyrin-apoprotein i n t e r a c t i o n i n the "T" s t a t e hemoglobin and myoglobin. Hoffman and a s s o c i a t e s (83-86) have shown that c o b a l t - s u b s t i t u t e d myoglobin and hemoglobin can combine r e v e r s i b l y and c o - o p e r a t i v e l y w i t h molecular oxygen, although, to a much l e s s e r extent than the n a t i v e p r o t e i n s . I t i s i n t e r e s t i n g to note i n Table 4.7 that although Co(II) o has a l a r g e r i o n i c radium (0.72A) than F e ( I I I ) , the out-of-plane d i s -placement of Co atom from the mean plane of the p y r r o l e nitrogens i s only o o 0.13A. In c o n t r a s t , the smaller F e ( I I I ) ( i o n i c radius = 0.64A) has a o o l a r g e r metal displacement ranging from 0.38A to 0.52A. Resonance Raman st u d i e s on CoHb and Hb by Woodruff et a l . , (87) i n d i c a t e that the Co(II) o atom out-of-plane displacement i n Co-Hb cannot exceed 0.2A. The a l l o s t e r i c model by Perutz on the mechanism of r e v e r s i b l e oxygen-binding c o - o p e r a t i v i t y i n hemoglobin depends d i r e c t l y on the l a r g e change on the P p . . . N e distance upon oxygenation (See Section 2.2). I t i s thus l o g i c a l to wonder whether -59-the Perutz's " t r i g g e r " mechanism i s c o n s i s t e n t w i t h the much smaller out-of-plane displacement of Co(II) i n model metalloporphyrins and CoHb. The most reasonable r a t i o n a l i s a t i o n of t h i s dilemma i s that of Hoard and Scheldt (64) who propose that the t e n s i o n w i t h i n the p r o t e i n i n deoxy-CoHb causes the porphinato core to undergo a s u b s t a n t i a l o doming of MJ.5A towards the Co atom. The doming r a i s e s the Co atom o o out-of-plane displacement to ^0.70A compatible to Fe atom (0.75A) i n o horse deoxy-Hb and 0.6A i n human deoxy-Hb. A support f o r the Hoard and Scheidt proposal (64) comes from the f a c t that the prophyrin s k e l e t o n i s r e a d i l y deformed normal to the mean plane. As one example, Ni(OEP) c r y s t a l l i s e s i n both a t r i n c l i n c i c form and a t e t r a g o n a l form (35). Both c r y s t a l s are v i r t u a l l y isomorphous, but yet the t e t r a g o n a l form e x h i b i t s marked n o n - p l a n a r i t y of the porphinato core while the t r i c l i n i c form i s e s s e n t i a - l y planar. CoHb i s t h e r e f o r e thought to achieve the "tense" quaternary s t r u c t u r e , not so much by the separation between the Co(II) atom and the mean porphyrin plane, but r a t h e r by marked "doming" of the porphyrin s k e l e t o n i t s e l f to move the c o b a l t nearer to the heme-linked h i s t i d i n e (F8). The "R" s t a t e quarternary s t r u c t u r e s of CoHb and FeHb ( i . e . , a f t e r oxygen-binding, so that the metal atom l i e s i n the porphyrin plane) are expected to be s i m i l a r , w h i l e the o v e r a l l f u n c t i o n a l p r o p e r t i e s are very d i f f e r e n t . For example the o x y g e n - a f f i r i t y of Co-Hb i s reduced by 10-100 times r e l a t i v e to n a t i v e Hb and Mb (86). Furthermore, Collman and a s s o c i a t e s (124) have shown that the f r e e energy d i f f e r e n c e between the i n t r i n s i c b i n d i n g of the f i r s t and f o u r t h oxygen to CoHb i s o n e - t h i r d that of Hb. Therefore, i t seems reasonable to conclude that the "T" s t a t e quarter-nary s t r u c t u r e s of CoHb and Hb must be a p p r e c i a b l y d i - f e r e n t , most probably because of the much smaller displacement of Co atom above the porphyrin -60-plane. In seeking a metalloporphyrin which, when r e c o n s t i t u t e d i n t o apo-hemoglobin, w i l l produce a "tense" conformation more s i m i l a r to that f o r the n a t i v e i r o n - c o n t a i n i n g p r o t e i n , one there f o r e seeks a case f o r which the metal displacement above the porphyrin plane i s more s i m i l a r to that f o r the corresponding h i g h - s p i n F e ( I I ) complex. From Table 4.7, i t i s c l e a r that there are r e l a t i v e l y few a t t r a c t i v e candidates. Mn-Hb has been prepared (80), and Moffat et a l . , (79) concluded from t h e i r c r y s t a l l o g r a p h i c s t u d i e s on Mn-Hb that deoxy-Mn(II)Hb w i l l adopt the normal "T" quaternary s t r u c t u r e . However, Mn(II)-Hb i s i r r e v e r s i b l y o x i d i s e d to Mn(III)-Hb which has been shown to be a c l o s e s t r u c t u r a l analog of met-Hb (79). Mg and Zn (76), w h i l e presumably s t a b l e toward o x i d a t i o n , e x h i b i t r e l a t i v e l y s m all metal displacements above the porphyrin plane, and may thus not be ap p r e c i a b l y b e t t e r than Co i n t h i s respect. The l a r g e i o n s , T l , Hf, Zr, and B i , give displacements which may be too l a r g e (compared to Fe ) , and i n the cases of Hf and Zr give u n s u i t a b l e c o o r d i n a t i o n number. However, the present r e s u l t s suggest that an indium porphyrin may be an i d e a l candidate f o r inducing the "tense" conformation i n hemoglobin, without the com p l i c a t i o n of a u t o x i d a t i o n , oxygen-binding to the in d i u m - l a b e l l e d subunits, and wi t h the n e a r - i d e a l displacement of the metal atom from the mean porphyrin plane. Indium has a f u r t h e r advantage that i t has an isotope ("''"'""'"In) which i s s u i t a b l e f o r gamma-gamma coincidence measurements. Part two of t h i s t h e s i s d e s cribes the r e c o n s t i t u t i o n of InMPP IX i n t o apomyoglobin, and how gamma-gamma angular c o r r e l a t i o n measurements can give motional p r o p e r t i e s of the p r o t e i n . - 6 1 -4.2 Bond Lengths and Angles The bond lengths and angles of Table 4.5 show that the square pyramidal InTPP:Cl molecule has very c l o s e to C ^ symmetry, as f a r as the I n , C l , and porphyrin skeleton atoms are concerned. (The phenyl t i l t s o are discussed i n S e c t i o n 4.4). The average In-N d i s t a n c e i s 2.156(6)A, which i s s u b s t a n t i a l l y l a r g e r than f o r the corresponding h i g h - s p i n F e ( I I I ) o complexes (Ca. 2.06-2.09A). This increase i s due p a r t l y to expansion of o the porphyrin core (radius of 2.067A f o r InTPPCl, compared to approximately 2.00 to 2.04 f o r i r o n ( I I I ) ) , and p a r t l y to an i n t r i n s i c a l l y longer In-N bond compared to Fe-N bond. However, the Fe-N bond l e n g t h f o r i r o n i n hemoglobin i s expected to increase to approximately the present In-N v a l u e , due to t e n s i o n produced by the proximal h i s t i d i n e l i g a n d of Hb (64,65,55); thus the indium porphyrin may s t i l l be a c l o s e s t r u c t u r a l analog f o r F e ( I I ) i n deoxyhemoglobin, even though the i s o l a t e d porphyrin s t r u c t u r e s appear r a t h e r d i f f e r e n t w i t h respect to metal-nitrogen bond len g t h s . - 6 2 -4.3 Doming of the Porphyrin Skeleton o The M-Np d i s t a n c e of 2.01A appears to be a nearly optimum value f o r minimal s t r a i n and u n d i s t o r t e d accomodation of the metal atom w i t h i n the c e n t r a l "hole" of the porphyrin. S t r u c t u r a l s t u d i e s (21) have shown that f o r a h i g h l y expanded porphinato core, the most e f f i c i e n t non-planar conformation i s doming of the core (18,36). Hoard and coworkers (15) a l s o have demonstrated experimentally the high degree of f l e x i b i l i t y of the porphyrin s k e l e t o n i n m e t a l l o p o r p h y r i n s t r u c t u r e s . The long o ( I n - N p ) a v d i s t a n c e , 2.156A which i s undoubtedly r e s p o n s i b l e f o r the s i g n i f i c a n t "doming" of the porphinato core i s c l e a r l y i l l u s t r a t e d i n the stereo-drawing i n Figure 4.3. I t i s evident from Figure 4.3 that InTPP:Cl i s non-planar, and e x h i b i t s a marked d e v i a t i o n s from p l a n a r i t y of the porphyrin atoms. Atomic displacements from the mean porphyrin plane of InTPP:Cl are i l l u s t r a t e d i n Figure 4.4. The perpendicular displacements ( p o s i t i v e and negative s i g n correspond to displacement above ( i . e . , toward the indium) or below the plane) from the mean plane of a l l 28 core porphyrin atoms i n Figure 4.4 show that the porphyrin s k e l e t o n i s appreciably "domed" upward toward the indium. The plane of o the four p y r r o l e n i t r o g e n atoms i s i n f a c t d i s p l a c e d an average of 0.1A above the mean plane of the core of the porphyrin atoms. The outermost p y r r o l e o r i n g carbons are depressed by up to 0.214A below the plane. o I t i s i n t e r e s t i n g to note that the doming i n 2-MeImFe(II)TPP i s 0.13A o w h i l e i t i s 0.1A f o r InTPPrCl. This suggests that the porphyrin skeleton i s not s i g n i f i c a n t l y d i s t o r t e d by the presence of indium atom. Fermi (74) pointed out that the doming of the porphinato core i n human deoxy-Hb i s o u n l i k e l y to exceed the value of 0.17 to 0.22A. The degree of doming i n InTPPrCl i s w e l l w i t h i n the range observed f o r high-spin f e r r i c porphyrins - 6 3 -(Table 4.7). I t i s expected the i n f l u e n c e by the p r o t e i n environment may increase the doming of indium porphinato core compatible to that i n n a t i v e hemoglobin. _ 82 — — 5 51 F i g u r e 4 . 4 . A t o m i c d i s p l a c e m e n t s ( A x 10 ) f r o m t h e mean p o r p h y r i n p l a n e ( p l a n e #2 o f T a b l e 4 . 6 ) o f I n T P P : C l . P o s i t i v e n u m b e r s c o r r e s p o n d t o d i s p l a c e m e n t s t o w a r d t h e i n d i u m a t o m . -65-4.4 Phenyl Rings: Crystal Packing A stereo view of a unit c e l l i s shown i n Figure 4.2, and i l l u s t r a t e s the moelcular packing. The closest nonbonded intermolecular contact o i s 2.48A between H4C4 and H3C3 at (1-x, 1-y, z ) , indicating that there i s no strong hydrogen bonding network and only the normal van der Waals forces between molecules. I t i s of interest to note that phenyl ring #1 i s close to an inversion center, and as a result i t i s twisted by only 66.17° with respect to the mean plane of the porphyrin skeleton, i n order to minmize the nonbonded contacts to i t s centrosymmetrically related image. The other three rings have dihedral angles with respect to the porphyrin mean plane which are closer to perpendicular (107.59°, 93.67°, and 98.33° for phenyl rings Z/2-//4, respectively). That the four phenyl rings are equivalent i n solution i s shown by the proton NMR spectrum of InTPPCl i n Figure 4.5. When th i s spectrum was f i r s t observed (107), the magnetically inequivalent ortho protons of the phenyl rings were incorrectly ascribed to "non-equivalence of phenyls i n pai r s " , presumably due to two chemically different sets of two phenyls each. The present c r y s t a l structure indicate that the inequivalence arises from the displacement of the indium above the porphyrin r i n g , making the upper (four) and lower (four) ortho protons inequivalent. The coalescence of these two ortho proton peaks on increase in temperature (125,126) must thus arise from internal rotation of the chemically equivalent phenyl groups about the bonds which connect the phenyl rings to the porphyrin. -66-I n T P P C I * d e b 1 1 10 F i g u r e 4 . 5 . ] H 100 MHz FT-NMR s p e c t r u m o f I n T P P : C l i n C D C 1 3 s o l v e n t . P e a k a s s i g n m e n t s a r e : ( a ) , r e s i d u a l C H C 1 3 (6 = 7 . 3 3 ) ; ( b ) , m- a n d p - p h e n y l p r o t o n s ( 7 . 8 7 ) ; ( c ) a n d ( d ) , o - p h e n y l p r o t o n s ( 8 . 2 0 a n d 8 . 4 3 ) ; a n d ( e ) , p y r r o l e g - p r o t o n s ( 9 . 1 5 ) . S p e c t r u m was o b t a i n e d u s i n g a V a r i a n X L - 1 0 0 FT-NMR s p e c t r o m e t e r , w i t h C D C U 2 u s e d f o r D l o c k , 10 t r a n s i e n t s , 6 . 0 s e c p u l s e d e l a y , a n d s e n s i t i v i t y e n h a n c e m e n t t i m e c o n s t a n t o f - l . o s e c . - 6 7 -Table 4 . 1 . C r y s t a l data and c o n d i t i o n s f o r data c o l l e c t i o n . C 4 4 H 2 8 C l I n N 4 Space Group: P2 1 a = 10.099(1) A b = 16.117(2) c = 21 .090(2) B = 9 0 . 7 0 ( 1 ) ° R a d i a t i o n : Scan t y p e : Scan r a n g e : Scan speed: A p e r t u r e : S t a n d a r d s : Data c o l l e c t e d : o ( I ) : /n Fw Vol Z Dc 763.015 3432.5 A 3 4 1.476 g cm - 3 D 0 a - 1 .48(1) g cm - 3 v = 7 . 2 2 cm" 1 Mo K a , g r a p h i t e monochromator u - 2e ( 0. 4 7 + 0 . 3 5 t a n e ) ° i n u, extended 25** f o r each background measurement 0 . 7 5 to 10.1 deg m i n " 1 , to g i v e I / o ( I ) ^ 2 0 1 .33 x 4 mm, 173 mm from c r y s t a l 190, 6 4 1 , 05T2, 604; measured every hour o f exposure time + h k i f o r 2e <_ 5 5 ° ; 7859 r e f l e c t i o n s { I n t + 4(DG) + (0.041 J 2 }* 5 ; Int i s i n t e g r a t e d peak c o u n t , BG i s the sum o f the background counts and 1 i s the i n t e n s i t y a by n e u t r a l buoyancy i n CCI4 and CH2C12 -68-T a b l e 4.2. F i n a l a t o m i c p o s i t i o n a l a n d t h e r m a l p a r a m e t e r s I t CW t T 1 o i ib 022 033 0 1 2 013 023 t a 3»93.1( 3) 1 7 9 7 . 0 ( 2) 1 5 1 6 . 3 ( 1) • . 2 3 ( 2 ) 3.3« (2) 2.6»(2) - 0 . 3 9 ( 2 ) • - 0 . 3 3 ( 1 ) 0 . 20 (1) C l 1 3 8 7 ( 1) 2388 ( D 1706 ( 1) S . 2 ( 1» 6 . 1( 1) 5 . 5 ( 1) 0 . 8 1 1) - 0 . 1 ( H - 0 . 5 ( 1) 1 ( 1 ) 328K ( •> • 99 ( 3) 17»9< 2) 5 . 6 ( 2J 3. • ( 2) 3 . 0 ( 2) - 0 . » ( 2) - 0 . 3 ( 2) 0 . 2 ( 2) • (•) 3522 ( •) 1369 { 3) S«B( 2) 5 . 5 ( 3 | 3 . 5 ( 2) 2 . 8 ( 2) - 0 . 3 ( 2) - 0 . 3 ( 2) 0 . 0 ( 2) • O ) »761( «) 2 7 3 9 ( 3) 1202 ( 2) 5 . 1 ( 2) • . 0 ( 2) 3 . 1 ( 21 - 0 . 7 ( 2) - 0 . 3 ( 2) 0 . 2 ( 2) 1 ( 2 ) • 5 7 9 1 • ) 1891 ( 3) 2«01( 2) « . « { 2) 3 . B ( 2) 3 . 1 ( 2) - 0 . 8 ( 2) - 0 . 6 ( 2) 0 . 5 ( 2) C ( D 28U» ( 5) - 1 1 9 ( 3) 13«1< 2) « . 8 ( 3) 3 . • ( 3) « . K 3) - 0 . 5 ( 2) - 0 . 3 ( 2 | - 0 . 0 ( 2) C ( 2 ) 2665 1 6) - 8 6 3 ( 3) 1703 ( 3) 6 . 9 1 •) 3 . 7 ( 3) • • * ( 3) - 1 . 0 ( 3) - 0 . 9 ( 3) 0 . 6 ( 2) C C D 3 0 i e ( 6) - 7 0 3 ( 3) 2 3 1 0 { 3) 7 . 0 ( •) • . 1 ( 3) • . 0 ( 3) - 0 . 8 ( 3) - 0 . 8 ( 3) 1 - 0 ( 2) C(«) 3 3 9 5 1 5) 150 ( 3) 2 3 U 6 ( 2) 5 . 2 ( 3) 3 . 7 ( 3) 3 . 2 ( 2) - 0 . 2 ( 2) - 0 . « ( 2) 0 . 5 ( 2) C (5 ) 3909 ( 5) 5 o 0 ( 3) 2886 < 2) • . 5 ( 3) 3 . 9 ( 3) 3 . 6 ( 2) - 0 . 6 ( 2) - 0 . 2 ( 2) 0 . 7 ( 2) C (6) tt u u 5 ( 5) 1 36 1 ( 3> 2909 ( 2) S . 0 ( 3) • • 1( 3) 3 . 1 ( 2) - 0 . 6 ( 2) - 0 . » ( 2) 0 . 6 ( 2) C(T) • 976 ( *) 1760 ( •) 3 * 6 2 ) 2) 6 . 7 ( 3) 5 . 1 ( 3) 3 . 1 ( 2) - 0 . 9 ( 3) - 1 . 0 ( 2) 0 . 9 ( 2) C ( 8 ) 5«23( 5) 2510 ( • ) 3286 ( 2) 5 . 8 ( 3) 5 . 5 ( 3) 3 . 5 { 3) - 1 . « ( 3) - 0 . 9 ( 2) 0.»< 2) C ( 9 ) 5199 ( 5) 263 1 ( 3) 2 6 1 9 ( 2) • . 3 ( 3) » . M 3) 3 . 2 ( 2) - 0 . & 1 2) - 0 . 7 ( 2) 0 . 2 ( 2) C ( 1 0 ) 5561 ( 5) 3277 ( 3) 2 2«7( 2) • . 7 ( 3) ».S< 3) 3 . 5 ( 2) - 0 . 7 ( 2) - 0 . 7 ( 2) O.u( 21 C ( U ) 5392 ( 5) 3 3 5 8 ( 3) 1590 ( 2) « . 9 ( 31 ».»( 3J 3 . 7 ( 2) - 1 . 2 1 2) - 0 . 5 ( 2) O . M 2) C ( 1 2 ) 5890 ( *) «02« ( •) 1207 { 2) 6 . 5 ( •) 5 . 1 ( 3) » . M 3) - 2 . 2 ( 3) - 0 . 7 ( 2) 0 . 6 ( 3) C ( 1 1 ) 5 5 7 5 ( 6) 3946 ( •) 5 9 7 ( 2) « . 1 ( 3) 5 . 2 ( 3) 3 . 9 ( 3) - 2 . 0 ( 3) - 0 . 1 ( 2) 0 . 9 ( 2) C{1«) • S S b ( 5) 3077 ( 3) 5 8 9 ( 2) » .7 { 3) « .0 ( 3) 3 . 1 ( 2) - 0 . « ( 2) 0 . 0 ( 2) 0 . 9 ( 2) C ( 1 5 | « 39 3 ( 5) 2667 ( 3) • 8 1 2) « . 7 ( 3) • . 3 ( 3) 3 . 1 ( 2) - 0 . 3 ( 2) 0 . 1( 2) O . M 2) C ( H | 3783 ( 5) 1885 ( 3) 33 ( 2) « .9 ( 3) « .0( 3) 3 . 1 ( 2) 0 . 1( 2) 0 . 0 ( 2) O . M 2) C ( 1 7 ) 3 3 9 0 ( *) 1«50 ( •) -S3»< 2) 6 . 9 ( «) • . » ( 3) 2 . 9 [ 2) - 0 . 1 ( 3) - 0 . 1 1 2) 0 . 3 ( 2) C<1S» 2 8 9 6 < M 7 1 1 ( 3) - 3 S S C 2) * .»< 3) 3) 3 . 1 ( 2) - 0 . 7 ( 2) - 0 . 5 ( 2) - O . M 2) 2966 ( 5) 6 6 3 ( 3) 32 1( 2 | S . 0 ( 3> 3 . 9 ( 3) 3 . 1 ( 2) - 0 . 2 ( 2) - 0 . 3 ( 2 | - O . M 2) C ( 2 0 | 2 6 6 5 1 5) - S O ( 3) 688 ( 2) « . 7 ( 3J ! - »< 3) 3 . 6 ( 2) - 0 . » ( 2) - 0 . 3 ( 2) - 0 . 7 1 2) « Est imated standard d e l a t i o n s In t h i s .nd other tab les a r t , 1 , e n In parentheses and correspond to the l e a s t j i g n U U a n t d t o U s . The p o s i t i o n a l par i n t e r s have been m u l t i p l i e d bjr 1 0 * and the thermal parameters bjr 1 0 * b U U • « i j / (2 . z i t*«j*) A 2 - The thermal e l l i p s o i d Is g'ven b / e x p [ - ( e , , n ? • ^ * 8,3' • Z ^ n k • 2e,3«i • 2e 2 j k i ) ] . -69-Table 4 . 3 . R i g i d group parameters. Group x c y c z c 6 c n 1C1 -2C1 -3C1 -4C1 -1C6 2C6 3C6 4C6 0.1566(2) 0.4074(3) 0.7040(3) 0.4835(3) - 0 . 1 4 0 2 ( 2 ) - 0 . 0 4 0 9 ( 2 ) 0.4590(2) 0.3297(2) -0.0041(1 ) 0.4037(1) 0.2875(1) -0.1213(1) 1 .430(3) - 1 . 6 4 2 ( 3 ) - 2 . 1 1 4 ( 3 ) - 1 . 8 9 9 ( 3 ) - 2 . 6 6 2 ( 2 ) 3.017(2) 2.997(3) - 3 . 1 0 0 ( 2 ) 0.666(3) - 2 . 1 3 7 ( 3 ) - 0 . 4 5 7 ( 3 ) 1 .245(3) Group R •> B l B 2 B3 * 4 B 5 B6 1 Cl -2C1 -3C1 -4C1 -1C6 2C6 3C6 4C6 3 . 3 8 ( 9 ) 3 . 3 8 ( 9 ) 3 . 37(9) 3 . 7 ( 1 ) 4 . 5 ( 1 ) 4 . 6 ( 1 ) i : i f ! l 5 . 3 ( 1 ) 5 . 1 ( 1 ) 4.9(1 6 . 3 ( 2 ) 4 . 8 ( 1 ) 5 . K D 5 . 2 ( 1 ) 6 . 2 ( 2 ) 4 . 7 ( 1 ) 5 . 0 ( 1 ) 5 . 6 ( 1 ) 6 . 3 ( 2 ) 4 . K D 4 . 3 ( 1 ) 4 . 7 ( 1 ) 5 . 5 ( 1 ) a See S . J . LaPlace and J . A. I b e r s , Acta C r y s t a l l o g r . 18, 511 (1965) f o r d e f i n i t i o n o f group parameters. k B n i s the temperature f a c t o r of atom C n i n the phenyl r i n g i n A^. Table 4 . 4 . Derived Hydrogen atom p o s i t i o n a l and thermal parameters 3 I t o i z y z O (A* ) BC ( 1 6 ) 2 5 5 30 - 6 3 5 . 9 H C ( 1 7 ) 3 4 6 1 6 5 - 9 6 6 . 2 B C ( 1 3 ) 5 7 9 4 1 7 24 6.3 H C ( 1 2 ) 6 3 5 4 5 0 1 3 6 6 .5 H C ( 8 ) 5 8 2 2 9 1 3 5 6 6 . 2 HC (7 ) 5 0 1 154 3 ea 6 . 2 H C (3) 3 0 2 - 1 0 9 2 6 5 6.3 H C ( 2 ) 2 3 5 - 1 3 9 154 6.3 H 1 C 2 2 8 - 7 0 7 0 7 . 0 B 1 C 3 - 6 4 - 1 7 9 11 6 .3 B 1 CU 6 4 - 2 4 9 - 6 4 7 . 3 B 1 C 5 2 8 5 - 2 1 0 - 7 9 7 . 2 B 1 C 6 3 7 7 - 1 0 1 - 1 9 6 . 5 H 4 C 2 2 7 6 3 4 9 - 7 5 6 . 8 H 4 C 3 3 1 2 3 9 2 - 1 8 0 7 . 5 B 4 C 4 5 1 9 3 7 3 - 2 2 6 7 . 9 B 4 C S 6 9 1 3 1 1 - 1 6 7 8 . 3 B 4 C 6 6 5 5 2 6 8 - 6 3 7 . 3 B 3 C 2 4 7 5 4 7 1 2 7 0 7 . 9 B 3 C 3 5 9 0 5 7 6 3 2 3 7 . 4 B 3 C 4 8 1 9 5 6 5 3 4 0 9 . 1 B 3 C 5 9 3 3 4 4 8 3 0 5 9 . 2 B 3 C 6 8 18 3 4 2 2 5 2 8 . 3 B 2 C 2 2 0 0 6 3 6 9 7 . 1 H 2 C 3 2 1 9 - 7 0 4 6 3 7 . 8 B 2 C 4 • 2 7 - 1 1 8 4 9 7 7 . 7 B 2 C 5 6 1 5 - 8 8 4 3 9 7 . 6 B 2 C 6 5 9 6 - 1 1 3 4 4 6 . 7 The p o s i t i o n a l parameters have been m u l t i p l i e d by 10 and the 2 thermal parameters by 10 . -71-Table 4 . 5 . S e l e c t e d I n t e r a t o m i c d i s t a n c e s ( A ) and angles (deg). In -In -In -In -In -N O ) N ( 2 ) N O ) N O ) N ( 2 ) N ( 2 ) N ( 3 ) N ( 3 ) N ( 4 ) N ( 4 ) C O ) C ( 2 ) C ( 3 ) C ( 4 ) N O ) N ( 2 ) N 3 N ( 4 ) C t - N ( 3 ) - N ( 4 ) - C ( l ) - C ( 4 ) - C ( 6 ) - C ( 9 ) - C ( l l ) - C ( 1 4 ) - C 1 6 ) - C ( 1 9 ) - C ( 3 - C ( 4 ) - C ( 5 ) 2 . 1 6 0 ( 4 ) D i s t a n c e s C ( 5 ) - C ( 6 ) 1 . 4 0 0 ( 7 ) 2 . 1 5 8 ( 4 ) C ( 6 ) - C ( 7 ) 1 . 4 3 0 ( 7 ) 2 . 1 5 8 ( 4 C ( 7 ) - C ( 8 ) 1 . 3 4 4 ( 7 ) 2 . 1 4 6 ( 4 ) C ( B ) - C ( 9 ) 1 . 4 3 1 ( 6 ) 2 . 3 6 9 ( 2 ) C ( 9 ) - C ( 1 0 ) 1 . 3 9 9 ( 7 ) 4 . 1 5 0 ( 6 ) C O O ) - C ( 1 1 ) 1 . 4 0 3 ( 7 ) 4 . 1 1 6 ( 5 ) Jill! 1 . 4 3 8 ( 7 ) 1 . 3 8 6 ( 6 ) - C ( 1 3 ) 1 . 3 5 4 ( 7 ) 1 . 3 8 3 ( 6 ) C ( 1 3 ) - C ( 1 4 ) 1 . 4 3 6 ( 7 ) 1 . 3 7 8 ( 6 C ( 1 4 ) - C ( 1 5 > 1 . 3 9 4 ( 7 ) 1 . 3 8 0 ( 6 ) C ( 1 5 ) - C ( 1 6 ) 1 . 4 0 3 ( 7 ) 1 . 3 8 0 ( 6 ) C ( 1 6 ) - C ( 1 7 ) 1 . 4 3 7 ( 7 ) 1 . 3 7 8 ( 6 ) C ( 1 7 ) - C ( 1 8 ) 1 . 3 4 4 ( 7 ) 1 . 3 7 7 ( 6 ) C 0 8 ) - C ( 1 9 ) 1 . 4 3 7 ( 6 ) 1 . 3 7 2 ( 6 ) C ( 1 9 ) - C ( 2 0 ) 1 . 4 1 2 ( 7 ) 1.393 ( 7 ) 1 . 4 3 4 ( 7 ) C ( 2 0 ) - C O ) 1 . 3 4 9 ( 7 ) C ( 2 0 ) - 1 C l 1 . 5 0 6 ( 6 ) 1 . 4 2 8 ( 7 ) C ( 5 ) - 2 C 1 1 . 4 9 £ ( 6 ) 1 . 4 1 1 ( 7 ) C O O ) - 3 C 1 1 . 5 0 2 ( 7 ) C 0 5 ) - 4 C 1 1 . 5 0 7 ( 6 ) N O ) N ( 2 ) N ( 3 ) N ( 4 ) N O ) N ( 2 ) C t C t C t C t In In In In In In In In N ( l ) N O ) N 2 ) N ( 2 ) N ( 3 ) N ( 3 ) N ( 4 ) N ( 4 ) N O ) urn N ( 2 ) N ( 2 ) N ( 3 ) N 3 *U) N M ) : : - C ( 1 6 ) -COB) 8 5 . 5 ( 2 ) 8 5 . 2 ( 1 ) 8 5 . 4 ( 2 ) 8 5 . 5 ( 2 ) 1 4 7 . 9 ( 2 ) 1 4 6 . 3 ( 2 ) 1 0 5 . 1 ( 1 ) 1 0 5 . 9 ( 1 ) 1 0 6 . 9 ( 1 ) 1 0 7 . 8 ( 1 ) 1 2 5 . 9 ( 3 ) 1 2 6 . 4 ( 3 ) 1 2 5 . 1 ( 3 ) 1 2 4 . 7 ( 3 ) 1 2 5 . 6 ( 3 ) 1 2 5 . 9 ( 3 ) 1 2 5 . 4 ( 3 ) 1 2 5 . 6 3 ) 1 2 5 . 7 ( 4 ) 1 2 4 . 7 ( 5 ) 1 2 5 . 9 4 ) 1 2 5 . 9 ( 4 1 2 5 . 5 4 1 2 5 . 5 ( 4 1 2 6 . 2 ( 4 1 2 5 . 8 ( 4 ) 1 0 8 . 2 ( 4 ) 1 0 8 . 9 4 1 0 8 . 4 ( 4 1 0 8 . 0 ( 4 1 0 9 . 0 ( 4 1 0 8 . 8 4 1 0 8 . 6 ( 4 ) 1 0 8 . 8 ( 4 ) Anql es 1 C 1 1 C 1 2 C 1 2 C 1 3 C 1 3 C 1 4 C 1 4 C 1 C O ) C ( 2 ) C ( 3 C ( 4 ) C ( 5 ) C ( 6 ) C ( 7 ) C ( 8 ) C ( 9 ) C O O ) C 0 3 ) 1 1 ) 1 ? ) 1 4 ) 1 5 ) C ( 1 6 ) C C ( 1 9 ) C ( 2 0 ) C ( 2 0 ) -C ( 2 0 ) -C ( 5 ) -C ( 5 ) -C ( 1 0 ) -C ( 1 0 ) -C 0 5 ) -C ( 1 5 ) -C ( 2 ) -C ( 3 ) -C ( 4 ) -C ( 5 ) -C ( 6 ) -C ( 7 ) -C ( 8 ) -C ( 9 ) -C O O ) -C O D -C ( 1 2 ) -C ( 1 3 ) -C ( 1 4 ) -C ( 1 5 ) -C ( 1 6 ) -C ( 1 7 ) • : ci!Si: : : C ( 1 9 ) C O ) C ( 4 ) C ( 6 ) C ( 9 ) C O D C ( 1 4 ) C ( 1 6 ) C ( 3 ) C ( 4 ) C ( 5 ) C ( 6 ) C ( 7 ) C ( 8 ) C ( 9 ) C ( 1 0 ) C O D C 1 2 ) C ( 1 3 ) C ( 1 4 ) C ( 1 5 ) C 1 6 ) C O D C ( 1 8 ) C ( 1 9 ) C ( 2 0 ) C O ) C ( 2 ) 1 1 5 . 5 ( 2 ) 1 1 8 . 4 ( 2 ) 1 1 6 . 1 ( 2 ) 1 1 7 . 0 ( 2 ) 1 1 6 . 3 ( 2 ) 1 1 7 . 4 ( 3 ) H E . 8 ( 2 ) 1 1 5 . 0 ( 2 ) 1 0 8 . 1 ( 5 ) 1 0 7 . 6 ( 4 ) 1 2 6 . 2 ( 4 ) 1 2 6 . 7 ( 4 ) 1 2 5 . 6 ( 4 ) 1 0 7 . 7 ( 4 ) 1 0 8 . 2 ( 5 ) 1 2 6 . 1 ( 5 ) 1 2 6 . 2 ( 5 ) 1 2 5 . 4 ( 5 ) 1 0 7 . 2 ( 5 ) 1 0 7 . 8 ( 4 ) 1 2 5 . 6 ( 4 ) 1 2 6 . 0 ( 4 ) 1 2 5 . 1 ( 4 ) 1 0 7 . 6 ( 4 ) 1 0 7 . 6 ( 4 ) 1 2 5 . 2 ( 4 ) 1 2 6 . 0 ( 4 ) 1 2 6 . 0 ( 5 ) -72-T a b l e 4.5, c o n t i n u e d . P y r r o l e 1 N - a 1.386(6) N - d 1.383(6) a - b 1.434(7) b - c 1 349(6) c - d 1.428 6 a - m 1.393(7) d - m 1.411(7) a - N - d N - a - b N - d - c a - b - c b - c - d 107.1(4) 108.2(4) 108.9(4) 108.1(5) 107.6(4) P y r r o l e 2 1.377(6) 1.378(6) 1.437(6) 1.344(7) 1.437(7) 1.403(7) 1.412(7) 107.3(1) 108.6(4) 108.8(4) 107.6(4) 107.6(4) P y r r o l e 3 1.380(6) 1.378(6) 1.438(7) 1.354(7) 1.436(7) 1.403(7) 1.394(7) 107.2(4) 109.0(4) 108.8(4) 107.2(5) 107.8(4) p y r r o l e 4 1.378(6) 1.380(6) 1.430(7) 1.344(7) 1.431 6) 1.400(7) 1.399(7) 107.7(4) 108.4(4) 108.0(4) 107.7(4) 108.2(5) A v e r a g e 1.380(4) 1.380(2) 1.435(4) 1.348(5) 1.433(4) 1 .402(7) 107.3(3) 108.5(3) 108.6(4) 107.6(4) 107.8(3) Table 4 . 6 . S e l e c t e d planes o f the p o r p h y r i n m a c r o c y c l i c s k e l e t o n Plane #1: - 0 . 8 9 8 3 x + 0.4132y + 0.1491z + 2.0711 = 0 a Plane #2: - 0 . 9 0 5 2 x + 0.4003y + 0.1426z + 2.2543 = 0 o P e r p e n d i c u l a r displacements (A) Plane #1 In 0.6104(3) C(10) c i . 2 . 978 (2) C O D N(D* 0 . 014 (4) C(12) N ( 2 ) * - 0 . 014 (4) C(13) N O ) * 0. 015 (4) C(14) N(4) - 0 . 014 (4) C(15) C ( D - 0 . .136 (5) C(16) C(2) - 0 . .347 (6) C(17) C(3) - 0 . .356 (6) C(18) C ( 4 ) - 0 . .117 (5) C(19) C(5) - 0 , .128 (5) C(20) C(6) - 0 .074 (5) 1C1 C(7) - 0 , .103 (6) 2C1 C(8) - 0 . 0 6 8 (6) 3C1 C(9) - 0 .029 (5) 4C1 - 0 . 0 5 2 ( 5 ) - 0 . 0 4 9 ( 5 ) - 0 . 1 8 5 ( 6 ) - 0 . 2 2 4 ( 6 ) - 0 . 0 8 6 ( 5 ) - 0 . 1 2 2 ( 5 ) - 0 . 0 9 5 ( 5 ) - 0 . 2 1 9 ( 6 ) - 0 . 2 0 4 ( 5 ) - 0 . 0 8 9 ( 5 ) - 0 . 1 4 1 ( 5 ) - 0 . 2 4 1 ( 3 ) - 0 . 3 2 7 ( 3 ) - 0 . 1 6 3 ( 3 ) - 0 . 3 1 0 ( 3 ) In Cl N O ) * N ( 2 ) * N ( 3 ) * N(4)=* C O ) * . C ( 2 ) * C ( 3 ) * C ( 4 ) * C ( 5 ) * C ( 6 ) * C ( 7 ) * C ( 8 ) * C ( 9 ) * Plane #2 0.7115(3) 3.079 (2) 0.141 0.066 0.091 0.108 0.012 -0.187 -0.211 0.007 (5) - 0 . 0 2 3 ( 3 ) 0.011 (5) - 0 . 0 3 8 (6) - 0 . 0 1 9 (6) 0.028 (5) (4) (4) (4) (4) (5) (6) (6) C ( 1 0 ) * - 0 . 0 0 6 (5) c ( n ) * 0 . 0 0 6 (5) C(12)* - 0 . 1 4 2 (6) C ( 1 3 ) * - 0 . 1 6 8 (6) C(14)* - 0 . 0 0 8 (5) C ( 1 5 ) * - 0 . 0 2 5 (5) C(16)* 0.023 (5) C(17)* - 0 . 0 8 2 (6) C ( 1 8 ) * -0.051 (6) C(19) 0.055 (5) C ( 2 0 ) * 0.015 (5) 1C1 -0.061 (3) 2C1 - 0 . 2 2 0 (4) 3C1 -0.141 (4) 4C1 -0.214 (4) 3 The plane equations are i n terms o f orthogonal c o o r d i n a t e s i n A. * I n d i c a t e s the atoms i n c l u d e d i n the c a l c u l a t i o n of the mean p l a n e . Table 4.7. Representative metalloporphyrins with s i g n i f i c a n t out-of-plane displacement of the metal i o n . a Metal Oxidation State Ionic Radius b Metalloporphyrin Coordination Number Metal Ion Q Displacement (M-N) Core j ave Radius Doming Reference Bi III 0.96 A Bi(OEP) 5 1.09 A 2.32 A 2.047 A Zr IV 0.79 (0Ac) 2Zr(0EP) 8 1.02 2.268,2.259 2.024,2.014 Hf IV 0.78 (0Ac) 2Hf(0EP) 8 1.01 2.257 2.016 Fe II 0.74 deoxyhemoglobin (horse) (6) 0.75f T l III 0.95 ClTl(OEP) 5 0.69 2.212 2.10 In III 0.81 ClIn(TPP) 5 0.61 , 2.156 2.067 Fe II 0.74 deoxyhemoglobin (human) (6) 0.60(a);0.63(8) 2.1(aorB) 2.008 <o, (VO) ( I D OV(OEP) 4 0.54 2.101 2.030 Mn III 0.66 (l-Melm)Mn(TPP) 5 0.52 2.128 2.065 Fe III 0.64 0[Fe(TPP)] 2 5 0.50 2.087 2.027 Fe III 0.64 ClFe(proto-IX) 5 0.48 2.062 2.007 Fe II 0.74 (2-MeIm)Fe(TPP) 5 0.42 2.086 2.044 Fe III 0.64 (2-MeIm)Fe(TpivPP) 5 0.40 2.072 2.033 Fe III 0.64 ClFe(TPP) 5 0.38 2.049 Zn II 0.74 (C10<,)Zn(TPP) 5 0.35 2.076 2.046 Zn II 0.74 (py)Zn(TPyP) 5 0.33 2.073 2.047 Zn II 0.74 (py)Zn(OEP) 5 0.31 2.067 2.043 Fe III 0.64 methemoglobin (horse) (6) M).3 f Fe II 0.74 deoxy-erythrocruorin (6) M).3 f Fe III 0.64 metmyoglobin (whale) (6) 0.27 2.04 2.00*> Mn III 0.66 CLMn(TPP) 5 0.27 2.008 1.989 Mg II 0.66 (H20)Mg(TPP) 5 0.27 2.072 2.055 Mn III 0.66 N3Mn(TPP) 5 0.23 2.005 1.992 Co II 0.72 (l-Melm)Co(OEP) 5 0.13 1.955 1.950 Co II 0.72 (l-Melm)Co(TPP) 5 0.13 1.977 1.973 0.13 A 0.17,0.21 0.13 0.06 0.10 22(o);0.17 0.06 0.04 0.04 0.06 0.13 0.03 0.09 0.04 0.09 0.13 0.0 0.03 0.01 114 115 115 62 116 This work (8) 74 . 117 66 48 41 64,65 118 40 119 21 120 28 25 95 121 122 123 55 54 Abbreviations: OAc, acetate; Me, methyl; py, pyridine; OEP, octaethylporphyrin dianion; TPP, tetraphenylporphyrin dianion; proto-IX, protoporphyrin IX dianion; TpyP, tetra(4-pyridyl)porphyrin dianion. from reference 8. perpendicular displacement of the metal ion from the mean plane of the four pyrrole nitrogens. average distance from the center of the plane of the four pyrrole nitrogens to a pyrrole nitrogen. difference between the displacement of the metal from the mean plane of the four pyrrole nitrogens and the mean plane of the 24-atom porphyrin skeleton. displacement in t h i s case i s measured from the mean plane of the 24-atom porphyrin skeleton. g t h i s distance was contralned to t h i s value i n r e f i n i n g the atomic positions. -75-PART TWO PERTURBED ANGULAR CORRELATION STUDY ON MYOGLOBIN (Radioactive "'""'""'"In-labelled p o r p h y r i n / R e c o n s t i t u t i o n / x determination) c - 7 6 -CHAPTER V PERTURBED ANGULAR CORRELATIONS General I n t r o d u c t i o n During the past decade, s e v e r a l l a b e l l i n g techniques have been developed to determine the r o t a t i o n a l c o r r e l a t i o n times, i n t e r n a l motions, and conformational changes i n b i o l o g i c a l macromolecules. As one example, fluorescence d e p o l a r i z a t i o n measurements can provide a mea-sure of the r o t a t i o n a l c o r r e l a t i o n time of a small chromophore bound to a macromolecule (127). R o t a t i o n a l c o r r e l a t i o n times can a l s o be obtained from magnetic r e l a x a t i o n times (128), and from ESR line-shape a n a l y s i s (129) i s only i n very recent years that the method of perturbed angular cor-r e l a t i o n s (P.A.C.) has been u t i l i z e d as a l a b e l l i n g technique i n the study of b i o l o g i c a l macromolecules. Several p u b l i c a t i o n s have discussed how P.A.C. measurements can give motional and s t r u c t u r a l i n f o r m a t i o n about b i o l o g i c a l macromolecules (7-12). The i n f o r m a t i o n gained from P.A.C. measurements i s often s i m i l a r to that obtained i n nuclear and paramagnetic s t u d i e s but, i n some cases not obtainable i n any other way.NMR i s a r e l a t i v e l y an i n s e n s i t i v e technique, e s p e c i a l l y i n the case of very d i l u t e samples, which i s o f t e n the case f o r -4 -5 b i o l o g i c a l macromolecules (10 -10 M). This d i f f i c u l t y a r i s e s from the weakness of an n.m.r. s i g n a l compared with the background noise of the instruments used. For proton-n.m.r. of b i o l o g i c a l macromolecules i n which aqueous solv e n t s are r e q u i r e d , i t i s necessary to prepare the sample i n D2O r a t h e r than i n water. This i s to e l i m i n a t e the strong H^O-proton resonance that obscures most of the resonances of protons of i n t e r e s t . -77-Moreover, i n t e r p r e t a t i o n of n.m.r. spectra f o r l a r g e macromolecules i s r a r e l y simple. The fluorescence d e p o l a r i z a t i o n technique r e q u i r e s o p t i c a l transparency f o r operation which thus g r e a t l y l i m i t s i t s p o t e n t i a l use. Both n.m.r. and esr techniques demand expensive equip-ment f o r operation. The P.A.C. method has the advantage of being a p p l i c a b l e to s o l u t i o n s and s o l i d s which opens the p o s s i b i l i t y f o r i n v i v o experimentation. The s i m p l i c i t y of experimental measurements, -12 w i t h c o n c e n t r a t i o n s e n s i t i v i t y approaching 10 M, together w i t h the f a c t that the t h e o r e t i c a l understanding of the e f f e c t s of molecular motion on angular c o r r e l a t i o n s has now become more complete, make P.A.C. a p o t e n t i a l l y u s e f u l l a b e l l i n g technique. Despite i t s obvious p o t e n t i a l , r e l a t i v e l y few P.A.C. s t u d i e s have been reported. The paucity of data i s probably a t t r i b u t a b l e to the l a c k of v e r s a t i l i t y i n s e l e c t i v e a t t a c h -ment of r a d i o a c t i v e r o t a t i o n a l l a b e l s to s p e c i f i c s i t e s on macromolecules. This t h e s i s demonstrates that "^"'"In-labelled porphyrins can be very s e l e c t i v e l y incorporated i n t o myoglobin. P.A.C. experiments r e q u i r e the i n c o r p o r a t i o n of a gamma emitter i n t o the molecules under study. Several isotopes (gamma emitters) can be used: l l l m C d ( t 1 =49 min), 6 2 Z n ( t , = 9 h ) , 1 1 9 m H g ( t ] =43 min), 2° 4Pb(t,=68 min), and 'i " 2 -3 "5 ^ ^ I n ( t , =2.8 days). I t i s obvious that ^"^In w i t h i t s convenient h a l f -'s l i f e , 2.8 days i s the u s e f u l isotope from the p r a c t i c a l viewpoint. Furthermore, i n d i u m - I l l produces two gamma rays i n succession, each w i t h a convenient energy f o r d e t e c t i o n . M a r s h a l l et a l . , (13,14) were the f i r s t to show how gamma-gamma coincidence measurements f o r t h i s type of energy cascade can give d i r e c t information about chemical bonding and motional f l e x i b i l i t y at an in d i u m - l a b e l l e d s i t e on a macromolecule. The r a d i o a c t i v e l a b e l s can be made s p e c i f i c by bindin g the r a d i o a c t i v e -78-n u c l e i f i r s t to some chemical complexes. For instance, the Cd isotope can be bound to a chemical complexing agent such as EDTA which i s covalently attached to an act i v e group such as a s u l f y d r y l reagent. This part of the thesis describes the preparation of indium-Ill l a b e l l e d meso-protoporphyrin IX, and how P.A.C. measurements can give r o t a t i o n a l c o r r e l a t i o n time of myoglobin. The present r e c o n s t i t u t i o n of indium-Ill meso-protoporphyrin IX into apomyoglobin (myoglobin with i t s native heme removed) represents the f i r s t motional probe located at the metal center of the active s i t e on a protein. The r o t a t i o n a l c o r r e l a t i o n time of myoglobin obtained by P.A.C. method i s 16 nsec at 12°C. The discrepancy between the experimental T^* and the calculated from Debye's model i s probably due to the non-spherical conformation of myoglobin. * x i s used to denote the r o t a t i o n a l c o r r e l a t i o n time, c -79-CHAPTER VI THEORY OF PERTURBED ANGULAR CORRELATIONS OF NUCLEAR RADIATION 6.1 Introduct ion For decades p h y s i c i s t s have employed the P . A . C . technique for the determination of proper t i e s of exci ted nuclear states and m u l t i p o l a r i t i e s of r a d i a t i o n s emitted, and of i n t e r a c t i o n s respons ib le for the emission. It i s only w i t h i n t h i s decade that chemists began to use P . A . C . measure-ments to obta in motional and s t r u c t u r a l information on b i o l o g i c a l macromolecules. The p r o b a b i l i t y W(6) for emission of a nuclear r a d i a t i o n depends on the angle 8 between the emitted r a d i a t i o n and some f ixed d i r e c t i o n . The t o t a l r a d i a t i o n from a r a d i o a c t i v e sample i s i s o t r o p i c i f the nuclear spins are randomly or iented i n space s ince there i s no preferred d i r e c t i o n for the emission of the r a d i a t i o n . An a n i s o t r o p i c pat tern of emission can be observed only from an ensemble of n u c l e i that are not randomly o r i e n t e d . One method of obta in ing or iented n u c l e i i s by coo l ing the nuclear sp in system to a very low temperature. At thermal e q u i l i b r i u m , a net o r i e n t a t i o n of the spin-system occurs with the d i s t r i b u t i o n of spin o r i e n t a t i o n s according to the Boltzman f u n c t i o n . A p p l i c a t i o n of a very s trong magnetic f i e l d or an e l e c t r i c f i e l d gradient w i l l a l so a l i g n the s p i n s . Another method i s the s e l e c t i o n of n u c l e i with a l igned spins as i n the case o f P . A . C . I f the n u c l e i such as i n d i u m - I l l , decay through success ive emission o f two r a d i a t i o n s , choosing only those n u c l e i which emit the f i r s t r a d i a t i o n i n a given d i r e c t i o n i s equivalent to s e l e c t i n g n u c l e i whose spins are e s s e n t i a l l y a l igned i n that d i r e c t i o n . The succeeding second r a d i a t i o n then shows a d e f i n i t e angular dependence w i t h respect to the d i r e c t i o n of the f i r s t r a d i a t i o n . There i s a strong angular c o r r e l a t i o n between the d i r e c t i o n s of propagation of the gamma rays i n cascade. I t i s t h i s property that enables the P.A.C. method to monitor molecular motion. Following the emission of the f i r s t gamma ray, the angular c o r r e l a t i o n , W ( 0 ) w i l l be s t r o n g l y perturbed i f the o r i e n t a t i o n of the s p i n of the nucleus i n the intermediate s t a t e changes by i n t e r a c t i o n w i t h i t s surroundings. In the s e m i - c l a s s i c a l p i c t u r e , these i n t e r a c t i o n s produce a preces s i o n of the n u c l e i around the symmetry a x i s . The change i n nuclear o r i e n t a t i o n produces an a l t e r e d angular c o r r e l a t i o n . Several workers (7,8,9) have observed that the angular c o r r e l a t i o n of the gamma ray cascade f o l l o w i n g the decay of ^ ^^In i s s t r o n g l y perturbed when the r a d i o a c t i v e i o n i s bound to a macromolecule i n aqueous s o l u t i o n . In order to observe an a n i s o t r o p i c c o r r e l a t i o n i n the absence of an a p p l i e d f i e l d , the i n t e r -mediate s t a t e much have nuclear spi n _> 1, so that the nucleus may possess a quadrupole moment. The quadrupolar i n t e r a c t i o n of the nuclear quad-rupole moment wi t h e x t e r n a l e l e c t r i c f i e l d gradient i s the b a s i s of the angular c o r r e l a t i o n . This i n t e r a c t i o n turns out to be an advantage f o r the study of molecular r o t a t i o n a l motion, because the nuclear s p i n o r i e n t a t i o n r a t e due to a quadrupolar i n t e r a c t i o n i s a f f e c t e d by molecular r o t a t i o n a l motion, but not by r e l a t i v e t r a n s l a t i o n a l motion. By the study of the perturbed angular c o r r e l a t i o n of gamma r a d i a t i o n from a r a p i d l y r e o r i e n t i n g r a d i o a c t i v e nucleus, the nuclear r e l a x a t i o n time can be measured. Since the nuclear r e l a x a t i o n time depends s t r o n g l y on the ra t e of r o t a t i o n of the molecule to which the r a d i o a c t i v e nucleus i s bound, i t may be used to estimate the r o t a t i o n a l c o r r e l a t i o n time, T c which i s a measure of the time i t takes f o r the molecule to change i t s o r i e n t a t i o n by the order of one r a d i a n . 6.2 T h e o r e t i c a l c o n s i d e r a t i o n The theory of angular c o r r e l a t i o n i s probably one of the best and most comprehensive t h e o r i e s on nuclear phenomena. For the present case, only the theory of extranuclear p e r t u r b a t i o n s on angular c o r r e l a t i o n s w i l l be considered. An e x c e l l e n t review of the theory of perturbed angular c o r r e l a t i o n s i s a v a i l a b l e i n the a r t i c l e by Frauenfelder and S t e f f e n (130). I t i s f e l t that a b r i e f d i s c u s s i o n of fluorescence d e p o l a r i z a t i o n w i l l provide a b e t t e r understanding to P.A.C. because conceptually and in s t r u m e n t a l l y i t i s analogous to P.A.C. In fluorescence d e p o l a r i z a t i o n , i f the e x c i t i n g r a d i a t i o n ( u s u a l l y i n the u l t r a - v i o l e t region) i s p o l a r i z e d , the p r o b a b i l i t y of absorption w i l l depend on the o r i e n t a t i o n of the molecule, being maximum i f the d i r e c t i o n of the d i p o l e moment change i s p a r a l l e l to the d i r e c t i o n of propagation. I f , w i t h i n the l i f e t i m e of the e x c i t e d e l e c t r o n i c s t a t e (^10 s e c ) , the molecules do not r o t a t e a p p r e c i a b l y , then the f l u o r e s c e n t r a d i a t i o n w i l l a l s o be h i g h l y p o l a r i z e d . In a s o l u t i o n of molecules, however, r o t a t i o n a l movement occurs. Since t h i s i s a random process, i t leads to randomization of the o r i e n t a t i o n of the d i p o l e moments i n the time between absorption and emission, which r e s u l t s i n d e p o l a r i z a t i o n of the f l u o r e s c e n t r a d i a t i o n . This means that fluorescence d e p o l a r i z a t i o n can be used to measure the r a t e of r o t a t i o n a l motion of the molecule c a r r y i n g the chromophore. I t must be noted that the use of fluorescence d e p o l a r i z a t i o n to measure can never be unambiguous because of the d i f f i c u l t y of separating i n t e r n a l r o t a t i o n of the chromophore from the r o t a t i o n of the molecule as a whole. In the case of P.A.C, both the -83-" p o l a r i z i n g " or a l i g n i n g of the nuclear spins and e m i t t i n g r a d i a t i o n come from the r a d i o a c t i v e sample whereas an i n i t i a l e x t e r n a l p o l a r i z i n g source i s required f o r the fluorescence measurements. Gamma-rays a r i s e from decay of e x c i t e d nuclear s t a t e s r a t h e r than e x c i t e d e l e c t r o n i c s t a t e s i n the case of fluorescence ( v i s i b l e r e g i o n ) . Consider an assembly of randomly o r i e n t e d n u c l e i i n which s t a t e A decays by successive emission of two gamma r a d i a t i o n s , y and Y2> t o the l e v e l s B and C, as shown i n Figure 6.1. The t o t a l r a d i a t i o n w i l l be i s o t r o p i c . The arrangement f o r the d i r e c t i o n a l c o r r e l a t i o n experiment i s i l l u s t r a t e d i n Figure 6.2. In the simplest case, the detector 1 i s designed to accept only r a d i a t i o n y^ which i s equivalent to choosing n u c l e i whose magnetic d i p o l e s (spins) are a l i g n e d according to that d i r e c t i o n . Detectors 2 and 3 are s e n s i t i v e only to r a d i a t i o n The detectors may count a l l photons that f a l l i n t h e i r s o l i d angles; however the coincidence analyzer s e l e c t s p r i n c i p a l l y only p a i r s of r a d i a t i o n s Y^ and y^ which are g e n e t i c a l l y r e l a t e d to each other. This i s accom-p l i s h e d by accepting a s i g n a l from d e t e c t o r 1 only i f a s i g n a l from de t e c t o r 2 a r r i v e s at the same time. In other words, only those r a d i a t i o n s which are emitted w i t h i n the r e s o l v i n g time, T of the coincidence c i r c u i t K are accepted. T y p i c a l values of T are 10 ^ to 10 ^ second. By proper K s e l e c t i o n of r e s o l v i n g time and source s t r e n g t h , the p o s s i b i l i t y of chance coincidence between unrelated r a d i a t i o n may be reduced to a t o l e r a b l e l e v e l . The angular d i s t r i b u t i o n of the second r a d i a t i o n Y2 w i t h respect to the f i r s t r a d i a t i o n y^ i s expressed as an angular c o r r e -l a t i o n f u n c t i o n , W(6,t) which i s a measure of the number of coincidences - 8 4 -A < \ \ \ Figure 6.1. Nuclear decay scheme of a level A which decays by the emiss ion of a radiat ion tfi into a l eve l B and then by the emission of a r a d i a t i o n 82 into a level C . -85-T i counter Tj counter Tj counter F i g u r e 6 . 2 . The a r r a n g e m e n t f o r t h e d i r e c t i o n a l c o r r e l a t i o n e x p e r i m e n t , a n d t h e o r i g i n o f a n i s o t r o p y i n d i r e c t i o n o f g a m m a - r a y e m i s s i o n f r o m i n i t i a l l y u n a l i g n e d n u c l e a r m a g n e t i c d i p o l e s . A l t h o u g h i n i t i a l m a g n e t i c d i p o l e d i r e c t i o n s a r e random ( u p p e r l e f t ) , r e s t r i c t i n g t h e d e t e c t i o n o f t h o s e r a y s t o a s i n g l e d i r e c t i o n s e l e c t s n u c l e i t h a t m u s t h a v e been a l i g n e d a c c o r d i n g t o t h a t e m i s s i o n d i r e c t i o n ( m i d d l e l e f t ) . Gamma r a y s s u b s e q u e n t l y e m i t t e d ( i . e . , y-|) f r o m t h o s e a l i g n e d n u c l e i w i l l be o b s e r v e d p r e f e r e n t i a l l y a t 1 8 0 ° r a t h e r t h a n 9 0 ° t o t h e d i r e c t i o n b e t w e e n t h e s a m p l e and t h e Y 2 " c o u n t e r ( r i g h t ) . F o r m o s t c a s e s , t h e e n e r g i e s o f Y ] a n d Y 2 a r e s u f f i c i e n t l y d i f f e r e n t t h a t t h e y a r e r e a d i l y d i s t i n g u i s a b l e by t h e d e t e c t o r s . -86-per u n i t time. By t h i s arrangement, an anisotropy between y^ a r r i v i n g a t detector 1 and a r r i v i n g at detectors 2 and 3 i s detected. I f the molecules are able to r o t a t e during the time between the emission of y^ and the observed y^, then the anisotropy w i l l be p a r t i a l l y l o s t due to the changing nuclear o r i e n t a t i o n of the nucleus i n the i n t e r -mediate s t a t e . For slow r o t a t i o n a l motion, the a n i s o t r o p y , A ( t ) w i l l decrease e x p o n e n t i a l l y w i t h time. E x p e r i m e n t a l l y , A ( t ) i s obtained from the expression below [ ( # Y 2 - c o u n t s at l S O ^ - C Z / y ^ c o u n t s at 90°)] A ( t ) = [iiy2 counts at 90°] ( 6 , 1 ) In gamma-gamma s t u d i e s , f a s t e r molecular r o t a t i o n u s u a l l y leads to slower exponential decay of anisotropy. I n d i u m - I l l decays by e l e c t r o n capture to cadmium-Ill followed by the successive emission of two gamma rays i n the 173-247keV cascade as shown i n Figure 6.3. For t h i s s o r t of energy cascade, S t e f f e n (131) showed that the coincidence counting r a t e , W(6,t) i s given by W(6,t) = f-e" t / TN[l+A P (cose)] (6.2) N where 6 i s the angle between the two gamma r a y s , P2(cos6) i s the Legendre 2 -7 polynomial l/2(3cos 6-1), T„(1.22x10 sec) i s the mean l i f e t i m e of the N intermediate nuclear s t a t e f o r the 247keV s t a t e of 1 : L 1 C d (132) , t i s the time i n t e r v a l between the emission of the two gamma ra y s , and * s a parameter that depends on the spins and m u l t i p o l a r i t i e s a s s o c i a t e d w i t h the cascade. The t h e o r e t i c a l value f o r the 173-24kKeV cascade, A22 = _0.18 (131). -87-Eneray Nuclear (kev) spin 420 397 7/2 "72 247 5/2 0 117 Cd t i 2.8 days t i = 1.2 x lcr ] 0 sec 173 150 kev, 247 11 = 49 min z ti - 8.5 x 10'8 sec z Stable F i g u r e 6 . 3 . The 1 7 3 - 2 4 7 keV g a m m a - r a y c a s c a d e o f ^ C d a f t e r t h e e l e c t r o n - c a p t u r e d e c a y o f ^ I n . -88-Following the emission of the f i r s t gamma, the angular c o r r e l a t i o n can be s t r o n g l y perturbed by i n t e r a c t i o n of the nuclear quadrupole moment 111 of Cd i n the metastable 247keV s t a t e with the f l u c t u a t i n g e l e c t r i c 111 f i e l d gradient at the Cd nucleus. The p e r t u r b a t i o n on the angular c o r r e l a t i o n can only be observed i f i s long enough and the nuclear moments of the intermediate s t a t e are la r g e enough. In t h i s case, the c o e f f i c i e n t of P2(cos6) i n Eq. 6.2 can be w r i t t e n as A 2 2 G 2 2 ( t ) where G22(t) i s an a t t e n t u a t i o n c o e f f i c i e n t (133). Eq. 6.2 can then be r e w r i t t e n as W(6,t) = j-e t / T N [ l + A 2 2 G 2 2 ( t , I , O D o , n , T c ) P 2 ( c o s 0 ) ] (6.3) N G„„(t,I,o) ,n , T ) i s the p e r t u r b a t i o n f a c t o r which represents the i n t e r -11 o c a c t i o n w i t h surroundings determined by the parameters 6eQV V -V % = h4i#-i) » n = -^H21 (the a8rTry zz parameter) and T , where Q i s the quadrupole moment of the intermediate s t a t e "^''"'"Cd, V , V , and V are components of the f i e l d gradient i n the p r i n c i p a l xx yy z z a x i s system, and T i s the r e l a x a t i o n time. I t i s obvious that the c informat i o n of p h y s i c a l i n t e r e s t i s contained i n the time-dependent p e r t u r b a t i o n f a c t o r , G 2 2 ( t ) . In the unperturbed case, G 2 2 ( t ) = l . - 8 9 -In the present s t u d i e s where "'""'"''"In-porphyrin complex i s bound to a macromolecule (myoglobin) i n aqueous s o l u t i o n , the angular c o r r e l a t i o n i s expected to be perturbed by the i n t e r a c t i o n of the nuclear quadrupole moment, Q of '''^Cd i n the intermediate s t a t e w i t h the f l u c t u a t i n g e l e c t r i c f i e l d gradients at the nucleus. The form of 0^2^) then depends on the dynamical d e t a i l s of the molecular motion. The approximate form of G 2 2 ( t ) may be d i s p l a y e d by a p l o t of the anisotropy = W ( T T,t)-W(7T/2,t) ( W(7T/2,t) 1 ~ 3 " 2 A 2 2 G 2 2 ( ' t ^ against the delay time, t between the emissions. In p r a c t i c e t i s the delay time between the two channels of the coincidence c i r c u i t . E x p e r i -mentally £"22(0 c a n be determined from the formula below r (r\ - - 2 - W ( T r , t ) - W O / 2 , t ) ( 6 5 ) G 2 2 U ; " A(t) W(Tr,t ) + 2 W(ir/ 2,t) The d e t a i l e d shape of the p l o t of G ^ C O v e r s u s delay time depends on the r e l a t i v e magnitude of the molecular r o t a t i o n a l c o r r e l a t i o n time, T of the 2 r a d i o a c t i v e probe, and upon the nuclear quadrupole i n t e r a c t i o n (e qQ) between the nuclear quadrupole moment (eQ) and the e l e c t r i c f i e l d (eq) at the nucleus i n the intermediate s t a t e . -90-6.2a Time-Dependent Quadrupole I n t e r a c t i o n - The L i m i t of Rapid Motion Time-dependent quadrupole i n t e r a c t i o n s represent the major p e r t u r b a t i o n f a c t o r on angular c o r r e l a t i o n s f o r the case i n l i q u i d s . This a r i s e s from the Brownian motion of the molecules i n a l i q u i d which give r i s e to r a p i d l y f l u c t u a t i n g e l e c t r i c f i e l d gradients that i n t e r a c t w i t h the nuclear e l e c t r i c quadrupole moment, eQ i n the intermediate s t a t e . Using time-perturbation theory, Abragam and Pound (133) have c a l c u l a t e d the e f f e c t of r a p i d molecular motion on the angular c o r r e l a t i o n (130,134). This i s the case where molecular r o t a t i o n a l c o r r e l a t i o n time, T i s short c compared to a period of the quadrupole frequency. As an example i s the s i t u a t i o n of a small molecule i n a non-viscous l i q u i d . For the r a p i d motion, G 2 2 ( t ) takes the form of a simple exponential f o r the case of a nuclear s p i n , I = 5/2, and an a x i a l l y symmetric e l e c t r i c f i e l d gradient (133) G 2 2 ( t ) = exp(-A 2t) (6.6) where h - i55o<« 2«®\/» ( 6 - 7 ) where double bar denotes an ensemble average, and T C i s the molecular c o r r e l a t i o n time. i s known as the r e l a x a t i o n constant. The above equations assume that the r o t a t i o n a l d i f f u s i o n i s i s o t r o p i c . The d i f f e r e n t i a l G 2 2 ( t ) i n Eq. 6.6 can be expressed i n the i n t e g r a l form G^F) = 1 /T N jTe" t / TNG 2 2(t)dt (6.8) - 9 1 -Combining Eqs. 6.6 and 6.8 gives G 2 2(~) = 1/(1+A 2T n) (6.9) The d i f f e r e n t i a l c o r r e l a t i o n Eq. 6.6 shows that G 2 2 ( t ) decreases monotonically w i t h time. In other words, a f t e r s u f f i c i e n t l y long time, the o r i g i n a l o r i e n t a t i o n of the assembly of n u c l e i i s completely l o s t . This i s a l s o r e f l e c t e d i n the i n t e g r a l c o r r e l a t i o n Eq. 6.9, which can become zero f o r l a r g e f i e l d strengths or long l i f e t i m e . The i n t e g r a l c o r r e l a t i o n i s easy to perform experimentally and i n much l e s s time than a measurement of d i f f e r e n t i a l G 2 2 ( t ) . However, some info r m a t i o n and d e t a i l s concerning the i n t e r a c t i o n mechanism i s u s u a l l y 111 l o s t i n the averaging process. In the present s t u d i e s on I n - l a b e l l e d myoglobin, t i m e - d i f f e r e n t i a l technique was used, which means the angular c o r r e l a t i o n i s measured as a f u n c t i o n of t , the delay time. From Eqs. 6.6 and 6.9, i t i s c l e a r that f o r l a r g e values of the a t t e n t u a t i o n f a c t o r vanishes and there i s no f i n i t e lower l i m i t . In other words, the angular c o r r e l a t i o n can be i s o t r o p i c f o r slow motion i n l i q u i d s . The r e l a x a t i o n constant, A 2 i s d i r e c t l y p r o p o r t i o n a l to the c o r r e l a t i o n time, T (Eq. 6.7) which i s u s u a l l y small f o r small molecules i n d i l u t e s o l u t i o n s : T c ~ 1 0 "''"'"sec. Consequently, as evident from Eqs. 6.6 and 6.7 that even f o r l a r g e quadrupole i n t e r a c t i o n , the a t t e n t u a t i o n of the angular c o r r e l a t i o n f o r s m all molecules i n l i q u i d s i s s m a l l . This e x p l a i n s why the anisotropy of angular c o r r e l a t i o n s remains l a r g e l y unperturbed f o r s m a l l molecules or f r e e ions i n d i l u t e s o l u t i o n s . The present study shows that s i m i l a r anisotropy pattern i s observed f o r "'""'^IriMPPIX and InTPP i n organic s o l v e n t s . -92-Based on the Debye model (135) f o r p o l a r l i q u i d s , T c i s given by where R i s the e f f e c t i v e molecular r a d i u s , £ i s the v i s c o s i t y , T i s the absolute temperature, and k i s the Boltzman's constant. Eq. 6.10 c l e a r l y shows that x ^ i s d i r e c t l y p r o p o r t i o n a l to the v i s c o s i t y of the l i q u i d (135,136). Consequently, changing the v i s c o s i t y w i l l a f f e c t the anisotropy of the angular c o r r e l a t i o n s . Measurements of t i m e - d i f f e r e n t i a l angular c o r r e l a t i o n s (TDAC) on aqueous s o l u t i o n s of 1 ' 1 ' 1 I n - C l ^ f o r v a r i o u s v i s c o s i t i e s have been performed by S t e f f e n (131). The r e s u l t s are shown i n Figure 6.4. As the molecular r o t a t i o n decreases due to increase i n s o l u t i o n v i s c o s i t y , the anisotropy decreases very r a p i d l y as expected from Eq. 6.9. The dependence of the ani s o t r o p y on the v i s c o s i t y i s c l e a r l y i l l u s -t r a t e d by a p l o t of G^^) versus v i s c o s i t y (137) i n Figure 6.5. The s o l i d l i n e represents the t h e o r e t i c a l curve c a l c u l a t e d on the assumption that T^ i s d i r e c t l y p r o p o r t i o n a l to the v i s c o s i t y of the l i q u i d . -93-0 100 2O0 300 400 500 DELAY l0( l«c ) ' F i g u r e 6 . 4 . A n i s o t r o p y f a c t o r ^^^-'^zZ °^ t n e Cd y-y d i r e c t i o n a l c o r r e l a t i o n as f u n c t i o n o f t h e d e l a y t i m e t f o r ^ I n s o u r c e s o f v a r i o u s v i s c o s i t i e s n . The s o u r c e i s a d i l u t e a q u e o u s s o l u t i o n i n I n C l ^ w h o s e v i s c o s i t y was v a r i e d by a d d i n g g l y c e r i n e . From S t e f f e n ( r e f . 1 3 1 ) . -94-F i g u r e 6 . 5 . The i n t e g r a l a n i s o t r o p y f a c t o r G2 2 (TO) A 2 2 o f t h e ^ C d Y-Y d i r e c t i o n a l c o r r e l a t i o n as f u n c t i o n o f t h e v i s c o s i t y n o f t h e ^ I n s o u r c e . From Hemmig and S t e f f e n ( r e f . 1 3 7 ) . -95-6.2b S t a t i c E l e c t r i c F i e l d Quadrupole I n t e r a c t i o n s - P o l y c r y s t a l l i n e Samples Although time-dependent quadrupole i n t e r a c t i o n s may occur i n c r y s t a l l i n e samples because of l a t t i c e v i b r a t i o n s and c r y s t a l d i s t o r t i o n s , s t a t i c e l e c t r i c f i e l d quadrupole i n t e r a c t i o n i s the p r i n c i p a l p e r t u r b a t i o n f a c t o r . Blume (134) considered the case of a p o l y c r y s t a l l i n e sample where there i s no molecular r o t a t i o n a l motion at a l l (x -*»). He showed that G„ 0(t) takes the form c l l G 2 2 ( t ) = (l/5)[l+(13/7)cosoo ot+(10/7)cos2a) t + (5/7)cos3o) 0t] (6.11) where (MQ = [3/21(21-1) ] (e 2qQ/fi) i s the quadrupole frequency. The d i f f e r e n t i a l c o r r e l a t i o n i n Eq. 6.11 i s a p e r i o d i c f u n c t i o n (a summation of cosines) i n c o n t r a s t to the case of time-dependent quadrupole i n t e r a c t i o n ( r a p i d motion) where G 2 2 ( t ) decreases e x p o n e n t i a l l y . In other words, the s t a t i c quadrupole i n t e r a c t i o n does not destroy the angular c o r r e l a t i o n s . Figure 6.6 shows the measurement made w i t h a m e t a l l i c ^"^In source (138). The f a c t that the experimental points l i e c l o s e l y on the t h e o r e t i c a l curve ( s o l i d l i n e c a l c u l a t e d according to the Eq. 6.11) provides evidence t h a t , to a good approximation, only s t a t i c e l e c t r i c quadrupole i n t e r a c t i o n i s present i n t h i s p o l y c r y s t a l l i n e indium metal source. In a p o l y c r y s t a l l i n e sample, the angular c o r r e l a t i o n i s never wiped out completely because the s t a t i c ensemble average over a l l e m i t t i n g nuclear o r i e n t a t i o n s gives a non-zero r e s u l t . However, i n the case of the time-dependent i n t e r a c t i o n s , the angular c o r r e l a t i o n can be completely wiped out because the d i r e c t i o n of the f i e l d at each nucleus changes - 9 6 -F i g u r e 6 . 6 D i f f e r e n t i a l a n i s o t r o p y A ( t ) o f t h e ^ C d y-y d i r e c t i o n a l c o r r e l a t i o n m e a s u r e d w i t h a p o l y c r s t a l l i n e I n - m e t a l s o u r c e . From Lehmann and M i l l e r ( r e f . 1 3 8 ) . continuously i n a random manner during the i n t e r v a l between emission of and Y 2« Figures 6.7a and 6.7b show the anisotropy of ^ "^Cd gamma-gamma c o r r e l a t i o n d i s p l a y e d by p o l y c r y s t a l l i n e non-metallic In^O^ and In(OH)^ (138). The s o l i d l i n e s represent the t h e o r e t i c a l curves f o r an a x i a l l y symmetric s t a t i c quadrupole i n t e r a c t i o n . The experimental curves do not show the usual c h a r a c t e r i s t i c p e r i o d i c behavior of the d i f f e r e n t i a l anisotropy A ( t ) f o r s t a t i c quadrupole i n t e r a c t i o n s (Figures 6.7a and 6.7b) as opposed to Figure 6.6. The non-metallic —8 sources show f i r s t a r a p i d decay of the c o r r e l a t i o n w i t h i n about 10 se and then p o s s i b l y f l u c t u a t e around the low val u e s . The discrepancy i s due to the e f f e c t s a r i s i n g from the e x c i t e d e l e c t r o n s h e l l a f t e r the K capture (See Appendix C). - 9 8 -Att) I r i j O , S O U R C E 1 0 15 ( 0 ) 2 0 2 5 X I O '"sec t • In ( O H ) , S O U R C E X I O ' ' s e c (b) Figure 6 . 7 . D i f f e r e n t i a l anisotropy A ( t ) of the ^ C d Y - Y d i r e c t i o n a l c o r r e l a t i o n observed with sources of (a) I i ^ O . ^ and (b) In ( O H ) 3 > From Lehmann and M i l l e r ( r e f . 1 3 8 ) . _gg_ 6.2c Time-Dependent Quadrupole I n t e r a c t i o n - The L i m i t of Slow Motion In the l i m i t of slow molecular motion, w i t h the assumption of a x i a l l y symmetric e l e c t r i c f i e l d , M a r s h a l l and Meares (13) show that the expression f o r G 2 2 ^ t ^ i s obtained by m u l t i p l y i n g Eq. 6.11 by the d i f f u s i o n damping f a c t o r , e x p ( - t / x c ) , 3 G 0 0 ( t ) = e x p ( - t / r ) l a Cos(w t) (6.12) 22 c n=o n o where a are the c o e f f i c i e n t s i n Eq. 6.11, and n T = (1/6D J (6.13) c r o t where D i s the r o t a t i o n a l d i f f u s i o n constant f o r a s p h e r i c a l molecule, r o t The i n t e g r a l a t t e n t u a t i o n f a c t o r G 2 2 ( t ) i s expressed as G ^ W = (l/5x )[1/C + (13/7) ( - 2 ^ ) + (10/7) C ^ q ^ > o o + (5/7) ( F f ^ ) ] (6.14) o i n which C = [(1/T„ + 1/T )] and N c w = (3/20)e 2qQ ° "ti The t h e o r e t i c a l dependence of i n t e g r a l p e r t u r b a t i o n or a t t e n t u a t i o n f a c t o r G22^ ° n r o t a t i o n a l c o r r e l a t i o n time, T c f o r the l i m i t i n g cases (slow and f a s t motion) i s c l e a r l y i l l u s t r a t e d by a p l o t of G22^ versus l o g T c as shown i n Figure 6.8. For small molecules i n non-viscous s o l u t i o n s , T can be as small as 10 "''"'"sec or l e s s , w h i l e f o r many p r o t e i n s , c —8 T i s i n the range of 10 sec (13). -100-Experiments by B a r r e t t et a l . , (139) and t h e o r e t i c a l c a l c u l a t i o n s by Lynden-Bell (140) demonstrate that the value of G22^ v a r i e s smoothly and continuously between those two l i m i t i n g cases ( r a p i d and slow motions), as shown by the broken l i n e i n F i gure 6.8. For a c o r r e l a t i o n time, x much l a r g e r than the l i f e t i m e , T„ of c N the intermediate nuclear s t a t e , the p e r t u r b a t i o n mechanism can no longer be considered as time-dependent. In f a c t the environment of the nucleus w i l l then be s t a t i o n a r y during the nuclear l i f e t i m e and the r e s u l t i n g i n t e r a c t i o n i s a s t a t i c one, f o r which a minimum c o r r e l a t i o n anisotropy e x i s t s as shown i n Figure 6.8. In the r e gion where x^ i s of the order of magnitude of x^, the i n t e r a c t i o n i s described a p p r o p r i a t e l y n e i t h e r by time-dependent nor by s t a t i c i n t e r a c t i o n mechanism. M a r s h a l l et a l . , (14) used parameters a s s o c i a t e d w i t h the decay of l l l m ^ t Q g e n e r a t e t h e o r e t i c a l l y s e v e r a l p l o t s that i l l u s t r a t e the dependence of ^22^^ o n an&^-e °^ attachment of the l a b e l , molecular geometry or i n t e r n a l r o t a t i o n r a t e (Figures 6.9-6.11). As can be seen from Figure 6.9, the shape of the p l o t of G^C 0 0 ) versus x^ can depend s t r o n g l y on the angle at which the l a b e l i s attached w i t h respect to the symmetry axes of the o v e r a l l moelcular shape. Figure 6.10 shows how &22^°°^ v a r i e s according to the r a t e of i n t e r n a l r o t a t i o n and angle of attachment of the l a b e l . I t i s c l e a r from Figure 6.10 that very r a p i d i n t e r n a l r o t a t i o n increases the a t t e n t u a t i o n f a c t o r , ^22^°°^ because i t e f f e c t i v e l y reduces the magnitude of the quadrupolar i n t e r a c t i o n f o r a f i x e d angle of attachment. However, i t i s i n t e r e s t i n g to note that i n the s i t u a t i o n where both the i n t e r n a l r o t a t i o n of the l a b e l and r e -o r i e n t a t i o n of the molecule as a whole are slow, i n t e r n a l r o t a t i o n decreases •10T-Figure 6.8. Theoretical relationship between the integral perturbation factor G22(°°) and the rotational correlation time T -102--12 - 1 0 - 8 - 6 - 4 Log (T,0,| F i g u r e 6 . 9 . P l o t s o f i n t e g r a l a n i s o t r o p y , G 9 ? ( » ) v e r s u s 111 r o t a t i o n a l c o r r e l a t i o n t i m e , T r Q t ( l o g s c a l e ) f o r a Cd n u c l e u s a t t a c h e d a t an a n g l e , e , w i t h r e s p e c t t o t h e m a i n s y m m e t r y a x i s o f a p r o l a t e m o l e c u l a r e l l i p s o i d . I n t h e f a s t -m o t i o n l i m i t ( l e f t - h a n d s e t o f c u r v e s ) , t h e i n t e g r a l a n i s o t r o p y v a r i e s w i t h t h e a n g l e o f a t t a c h m e n t , 6 , i n t h e o r d e r 6 0 ° > 9 0°>30° > 0 ° . I n t h e s l o w - m o t i o n l i m i t ( r i g h t - h a n d s e t o f c u r v e s ) , t h e i n t e g r a l a n i s o t r o p y v a r i e s w i t h e i n t h e o r d e r 0 ° > 3 0 ° > 6 0 ° > 9 0 ° . From M a r s h a l l e t a l . , ( r e f . 14) - 1 0 3 -03 -12 -11 -IO -9 LOG (I/6D) F i g u r e 6 . 1 0 . P l o t s of the i n t e g r a l a n i s o t r o p y i n the f a s t - m o t i o n l i m i t versus 1og(6D), where D i s the r o t a t i o n a l d i f f u s i o n constant f o r a s p h e r i c a l molecule and i s the d i f f u s i o n c o n s t a n t f o r i n t e r n a l r o t a t i o n . Each f a m i l y of curves corresponds to a f i x e d c h o i c e of "attachment a n g l e " as l i s t e d on the f i g u r e . For i n d i v i d u a l curves (A) through ( D ) , the i n t e r n a l r o t a t i o n d i f f u s i o n c o n s t a n t D ^ I O 1 2 , 1 0 1 1 , 1 0 1 0 , and 10 9 s e c " 1 , r e s p e c t i v e l y . The lowest curve i n each set (dashed l i n e ) i s the i n t e g r a l a n i s o t r o p y i n the absence of i n t e r n a l r o t a t i o n , or e q u i v a l e n t f o r zero "attachment a n g l e " . From M a r s h a l l e t a l . , ( r e f . 14) . - 1 0 4 -L O G (6DM) F i g u r e 6 . 1 1 . P l o t s of i n t e g r a l a n i s o t r o p y versus l o g ( 6 D ^ ) , where i s the r o t a t i o n a l d i f f u s i o n constant f o r a s p h e r i c a l macromolecule { i n the slow-motion l i m i t ) . The two s e t curves correspond to the two c h o i c e s f o r the i n t e g r a l r o t a t i o n a l d i f f u s i o n constant shown on the f i g u r e . The i n t e g r a l a n i s o t r o p y v a r i e s w i t h the d i f f e r e n t angles o f attachment i n the o r d e r , 0 ° > 3 0 ° > 6 0 o > 9 0 o , f o r curves a - d , r e s p e c t i v e l y . The r e s u l t f o r e=0° i s the same as f o r no i n t e r n a l r o t a t i o n a t a l l . From M a r s h a l l et a l . , ( r e f . 14). -105-the ^22^^ values as i l l u s t r a t e d i n Figure 6.11. I t can be i n f e r r e d from Figure 6.11 that when the molecular r o t a t i o n i s slow, i n t e r n a l r o t a t i o n does not a f f e c t the observed &22^m^ a p p r e c i a b l y unless the r a t e of i n t e r n a l r o t a t i o n i s s i g n i f i c a n t l y f a s t e r than the r a t e of r o t a t i o n of the molecule as a whole. I t i s important to note that i n t e r n a l r o t a t i o n , whether f a s t or slow, has no e f f e c t on the angular c o r r e l a t i o n when the angle of attachment of the l a b e l i s zero degrees. The above d i s c u s s i o n c l e a r l y demonstrates the p o t e n t i a l of gamma-gamma c o r r e l a t i o n technique as a means to study s p e c i f i c s i t e s on l a r g e b i o l o g i c a l l y i n t e r e s t i n g macromolecules such as hemoglobin, cytochromes. Information l i k e the degree of i n t e r n a l f l e x i b i l i t y at the s i t e of attachment of the r a d i o a c t i v e t r a c e r to the macromoelcule i . e . the r a t e of i n t e r n a l motion, as w e l l as the geometry of the complex can be obtained. Although the same informat i o n i s a c c e s s i b l e by ESR technique and fluorescence d e p o l a r i z a t i o n , the gamma-gamma experiment o f f e r s the -12 advantages of r e q u i r i n g concentrations which can be as low as 10 M and the c a p a b i l i t y of i n v i v o experimentation. -106-6.2d F i e l d s Without A x i a l Symmetry The preceding d i s c u s s i o n s are r e s t r i c t e d to the cases where a x i a l l y symmetric e l e c t r i c f i e l d g radients (EFG) are assumed. The r e s u l t s f o r a x i a l l y symmetric EFG cases can be extended to the s i t u a t i o n s of non-a x i a l l y symmetric e l e c t r i c f i e l d s by i n t r o d u c i n g an asymmetry parameter, n to account f o r no n - a x i a l symmetry i n the quadrupole i n t e r a c t i o n s . Considers the case of slow molecular motion, Eq. 6.12 ( f o r a x i a l l y symmetric EFG) becomes 3 G„.(t) = exp(-t/T ) Z b (n)c o s [ f (n)o> t ] (6.15) Ll c n n o n=o where f and the c o e f f i c i e n t s b (n) can be evaluated (141). For the o n quadrupole frequencies u (n)=f (n)w are no longer harmonic. For n=o n n o ( a x i a l l y symmetric) f ^ , f ^ , and f ^ take on i n t e g r a l v a l u e s . Matthias et a l . , (142,143) have performed such c a l c u l a t i o n s on p o l y c r y s t a l l i n e sources. Figures 6.12 and 6.13 show the d i f f e r e n t i a l Q>^(t) and time - i n t e g r a t e d f a c t o r , G22^° 0^ ^ o r s P i n - v a l u e s I = 2 a n d 1=5/2 and f o r v a r i o u s values of the asymmetry parameter. 181 Figure 6.14 shows the r e s u l t f o r that Ta gamma-gamma cascade w i t h p o l y c r y s t a l l i n e H^-metal source (144). The s o l i d l i n e i s the t h e o r e t i c a l curve c a l c u l a t e d on the assumption of an a x i a l l y symmetric s t a t i c quadrupole i n t e r a c t i o n . I t i s c l e a r l y evident from Figure 6.14 that there i s a discrepancy between the experimental and t h e o r e t i c a l r e s u l t s . However, i f the experimental data are i n t e r p r e t e d on the b a s i s of n o n - a x i a l l y symmetric quadrupole i n t e r a c t i o n s together w i t h an i n c l u s i o n of an asymmetry parameter, n=0.5 i n t o the c a l c u l a t i o n , an e x c e l l e n t agreement with the experimental r e s u l t s i s obtained. As - 1 0 7 -1.0 | I.2| V-08 0 . 4 0.6 1 * \ ' ' 11 \ : /<\\ / / \ ; G„ 0.4 (t) 0 . 2 /'•• \\; '• i.j 1 \kZl/'/7'' : I T V / / « ' >\ / , V / / •' \ * 1 \ i i .) i- to 0 . ;  - 0 . 2 1 . . . . 1 . . . . i . . . . i . ...<... . 1 0.5 1.0 1.5 2 . 0 2.5 3 . 0 Ul.l/lT • I . . . . I . . . . I .. I . . . . i . . . . I o 3 ro Ts z'o 4.5 3 . 0 0 J . t / 7 T F i g u r e 6 . 1 2 . D i f f e r e n t i a l a t t e n t u a t i o n c o e f f i c i e n t s G 2 2 ( t ) f o r r h o m b i c q u a d r u p o l e i n t e r a c t i o n i n p o l y c r y s t a l l i n e s o u r c e s f o r i n t e r m e d i a t e s t a t e s p i n s 1=2 a n d 1 = 5 / 2 . The p a r a m e t e r n i s t h e a s y m e t r y p a r a m e t e r n = ( V x x — V y y ) / V z z . From M a t t h i a s e t a l . , ( r e f . 1 4 2 ) . -108--. F i g u r e 6.13. T i m e - i n t e g r a t e d a t t e n t u a t i o n c o e f f i c i e n t s G22(°°) f o r r h o m b i c q u a d r u p o l e i n t e r a c t i o n i n p o l y c r y s t a l l i n e s o u r c e s f o r s p i n s 1=2 and 1 = 5 / 2 . From M a t t h i a s e t a l . , ( r e f . 143). i -109= F i g u r e 6 . 1 4 . D i f f e r e n t i a l a n i s o t r o p y A ( t ) o f t h e Ta Y - Y d i r e c t i o n a l c o r r e l a t i o n m e a s u r e d w i t h a p o l y c r y s t a l l H f - m e t a l s o u r c e . From S c h e c t e r and S o m m e r f e l d t ( r e f . 1 4 4 ) . -110-a matter of f a c t , t h i s was the f i r s t i n d i c a t i o n of the presence of a n o n - a x i a l l y symmetric s t a t i c quadrupole p e r t u r b a t i o n on angular c o r r e l a t i o n s . I t i s c l e a r from the two examples that the strong dependence of anisotropy on n (asymmetry parameter) can be used to give i n f o r m a t i o n on the geometry of the molecules under study. CHAPTER VII APPLICATIONS OF GAMMA-GAMMA CORRELATIONS TO BIOLOGICAL MACROMOLECULES This chapter i s intended to i l l u s t r a t e b r i e f l y how P.A.C. measurements can be used to ob t a i n motional i n f o r m a t i o n on b i o l o g i c a l macromolecules, and f o r t h i s purpose two examples w i l l be discussed. Meares et a l . , (8) a p p l i e d d i r e c t i o n a l gamma-gamma c o r r e l a t i o n s f o r the study of carbonic anhydrase. The anisotropy of f r e e ^^mCd^+ i n s o l u t i o n decreases s l i g h t l y w i t h time as i l l u s t r a t e d i n Figure 7.1a. The weakly perturbed angular c o r r e l a t i o n i s as expected from Eqs. 6.6 2 and 6.7 sinc e both e qQ and x ^ are sm a l l . Even,in the presence of na t i v e carbonic anhydrase, the anisotropy p a t t e r n i s s i m i l a r to that of the f r e e ^^~mCd^+ except f o r a s l i g h t l y f a s t e r decay of the gamma-ray anisotropy (Figure 7.1b). This i s a t t r i b u t e d to the increase i n s o l u t i o n v i s c o s i t y by the a d d i t i o n of the n a t i v e enzyme. The increase i n s o l u t i o n v i s c o s i t y r e s u l t s i n a slower r o t a t i o n a l d i f f u s i o n of ^ ^ m C d . The angular c o r r e l a t i o n i s s t r o n g l y perturbed when apo-carbonic anhydrase (the n a t i v e 2+ enzyme w i t h the a c t i v e - s i t e Zn removed) was added (Figure 7.1c). In t h i s case, x c>(e^qQ) \ and sinc e the r o t a t i o n a l d i f f u s i o n of the ^ ""^ mCd^ +-l a b e l l e d p r o t e i n i s now much slower, the r a p i d decay of the anisotropy w i t h time i s co n s i s t e n t w i t h Eqs. 6.6 and 6.7. The next example considers the a p p l i c a t i o n of the P.A.C. technique f o r i n v i v o experimentation.Goodwin et a l . , (145) a p p l i e d P.A.C. technique to f o l l o w ^ ^ I n t r a c e r metabolism i n l i v e mice. Altogether s i x i n d i u m - l a b e l l e d complexes were i n j e c t e d i n t r a v e n o u s l y i n t o i n t a c t , l i v e Swiss Webster mice, and gamma-rays were counted e x t e r n a l l y . The r e s u l t s of the i n t e g r a l -112*9 0 . 2 0 . 1 0 . 0 H-- 0 . 1 5 0 1 0 0 t (nsec) (c) 1 5 0 F i g u r e 7 . 1 . A n i s o t r o p y o f c o r r e l a t e d g a m m a - r a y e m i s s i o n f r o m ^ 1 m C d + + i o n i n v a r i o u s c h e m i c a l e n v i r o n m e n t s , ( a ) f r e e ^ l l m C d + + i n s o l u t i o n ; (b) 1 1 1 m C d + + i n t h e p r e s e n c e o f 3 x 1 0 ' ^ M n a t i v e c a r b o n i c a n h y d r a s e ( i . e . , e n z y m e w i t h n o a v a i l a b l e C d + + - b i n d i n g s i t e s ) , a n d ( c ) l l l m C d + + i n t h e p r e s e n c e o f 2 . 5 x 10"4M a p o c a r b o n i c a n h y d r a s e ( I . e . , e n z y m e w i t h o n e s t r o n g C d + + - b i n d i n g s i t e a v a i l a b l e ) . F r o m M e a r e s e t a l . , ( r e f . 8). a t t e n t u a t i o n f a c t o r , G^O") w e r e tabulated i n Table 7.1. When """"InCJlg was i n j e c t e d i n t r a v e n o u s l y i n t o a l i v e mice, the low G^C 0 0 ) v a l u e i n d i c a t e s that ^"^In binds to t r a n s f e r r i n (Figure 7.2) which i s con-s i s t e n t w i t h the observation that indium binds to t r a n s f e r r i n almost q u a n t i t a t i v e l y (146,147). From Table 7.1, the low G22^°°^ values f o r I n - c i t r a t e and In-NTA a f t e r i n j e c t i o n i n d i c a t e that i n d i u m - I l l d i s -s o c i a t e d from the complexes and bound to the t r a n s f e r r i n . The low G ^ t 0 0 ) values r e f l e c t e d by the long r o t a t i o n a l c o r r e l a t i o n times (t ) are c h a r a c t e r i s t i c values f o r macromolecules (8,13). In c o n t r a s t , f o l l o w i n g the i n j e c t i o n of In-EDTA, In-PhDTA, and In-CyDTA, the i n t e g r a l measurements give high G^^) values which means these compounds are e s s e n t i a l l y i n t a c t i n the l i v e mice. These s t u d i e s by Goodwin et a l . , (145) c l e a r l y demonstrate the p o t e n t i a l of the P.A.C. technique i n determining the f a t e of "'""'""'"In-labelled t r a c e r s i n humans by the e x t e r n a l countings of the gamma-rays emitted by the r a d i o a c t i v e t r a c e r . There remains a few remarks concerning the s o r t of informat i o n that can be extracted from the shape of the p l o t of anisotropy versus delay time. As mentioned i n Se c t i o n 6.2c, the angle of attachment of the l a b e l s can be determined by a n a l y z i n g the t i m e - d i f f e r e n t i a l coincidence spectrum. I f the spectrum shows a s e r i e s of minima, the l a b e l l e d molecules are g e n e r a l l y o r i e n t e d i n the same d i r e c t i o n . The spacing between the minima w i l l be determined by the o r i e n t a t i o n of the p r i n c i p a l a x i s of an ordered array of molecules w i t h respect to the d i r e c t i o n of the f i r s t gamma emission. Tendon, f i b e r s , and membranes are examples of ordered arrays of molecules that may be studi e d by the P.A.C. method. The study of conformational changes i n polypeptide chains ( e s p e c i a l l y h e l i x -random c o i l t r a n s i t i o n s ) h a s been a subject of considerable i n t e r e s t . A Table 7.1. In Compounds Studied In Vivo Compound No. of Mice *PAC Anisotropy=}= Column Chromatography (%) Transferrin Compound RIEt Tr a n s f e r r i n Band I n - t r a n s f e r r i n 7 0.076±0.004 100 0 Po s i t i v e I n - c i t r a t e 4 0.081±0.004 100 0 Po s i t i v e In-NTA 3 0.080±0.005 100 0 Po s i t i v e In-o-PhDTA 3 0.122±0.007 9.8 90.2 Negative In-CyDTA 3 0.128±0.005 10.7 89.3 Negative In-EDTA 3 0.120±0.007 10.2 89.8 Negative * PAC = Perturbed Angular Co r r e l a t i o n t RIE = Radioimmunoelectrophoresis =j= Mean of A±2 S.D. From Goodwin et a l . , ( r e f . 145). - 1 1 5 -F i g u r e 7.2. I n v i v o a n g u l a r c o r r e l a t i o n s o f g a m m a - r a y s f o l l o w i n g i n t r a v e n o u s i n j e c t i o n o f t h r e e l a b e l l e d i n d i u m compounds i n t o mice. I n i t i a l l o w v a l u e s f o r c i t r a t e and c h l o r i d e i n d i c a t e p r o t e i n-binding. I n - E D T A i s n o t p r o t e i n b o u n d . From G o o d w i n e t a l . , ( r e f . 1 4 5 ) . -116-f a i r l y r i g i d h e l i c a l conformation i s expected to give a coincidence spec-trum w i t h a s i n g l e minimum. However, upon t r a n s i t i o n to a random c o i l e f f e c t e d by pH change, the increase i n f l e x i b i l i t y of the chain i s r e f l e c t e d by an exponential decay curve c h a r a c t e r i s t i c of f r e e mole-cules i n s o l u t i o n . This i s i l l u s t r a t e d i n Figure 7.3 i n which M a r t i n et a l . , (148) studi e d the helix-random c o i l t r a n s i t i o n i n glutamic a c i d by P.A.C. Since P.A.C. i s a p p l i c a b l e to s o l i d s , i t means surface l a y e r s , powders, composite m a t e r i a l s e t c . , can a l s o be s t u d i e d . P.A.C. can a l s o be used to determine the s i z e of a c l e f t i n a macromolecule by a t t a c h i n g an appropriate r o t a t i o n a l t r a c e r of known s i z e to the c l e f t . I f a small r o t a t i o n a l t r a c e r i s incorporated i n t o the c l e f t , the greater movement of the t r a c e r i s r e f l e c t e d by an exponential decay i n the t i m e - d i f f e r e n t i a l coincidence spectrum. On the other hand, i f a s i n g l e minimum occurs, the r o t a t i o n a l t r a c e r i s r i g i d l y attached to the molecule and moves with a r o t a t i o n a l c o r r e l a t i o n time that i s long compared to the quadrupolar i n t e r a c t i o n . 10 20 30 CHANNEL NUMBER F i g u r e 7.3. T i m e - d i f f e r e n t i a l perturbed angular c o r r e l a t i o n (TDPAC) s p e c t r a f o r samples of p o l y g l u t a m i c a c i d a t pH=4.0 (upper curve) and pH=7.8 (lower c u r v e ) . The upper curve corresponds to the t i g h t h e l i x conformation whereas the lower curve i s f o r the random c o i l c o n f i g u r a t i o n . - 1 1 8 -CHAPTER V I I I EXPERIMENTAL WORK 8.1 P r e p a r a t i o n of Meso-protoporphyrin IX Meso-protoporphyrin IX (MPP IX) was chosen f o r the present study. This porphyrin i s more s t a b l e than protoporphyrin IX because the v i n y l groups of the protoporphyrin IX are chemically and photochemically l a b i l e . As an example, when a s o l u t i o n of protoporphyrin IX i n benzene or p y r i d i n e i s exposed to l i g h t , an o x i d a t i o n product known as photo-pr o t o p h y r i n IX i s formed (149). However, hydrogenation of the v i n y l groups i s accompanied by an undesirable r e d u c t i o n i n aqueous s o l u b i l i t y of the r e s u l t i n g meso-protoporphyrin IX. M a t e r i a l s : F e r r i - p r o t o p o r p h y r i n IX was obtained from Strem Chemicals, Inc.; palladium oxide (PdO. x ^ 0 ) was from MCB; formic a c i d (90%) was obtained from F i s h e r ; ammonia from A l l i e d Chemical Canada, L t d . ; ammonium acetate from MCB; sodium t a r t r a t e AR was obtained from M a l l i n c k r o d t . Procedure: The procedure f o r the prep a r a t i o n of meso-protoporphyrin IX from f e r r i - p r o t o p o r p h y r i n IX was e s s e n t i a l l y that of Taylor (150). The method involved the simultaneous r e d u c t i o n of the v i n y l sidechains to e t h y l groups and removal of i r o n through the a c t i o n of formic a c i d . The palladium oxide was used as the reducing c a t a l y s t . Ferro-proto-porphyrin IX was i n i t i a l l y formed by the r e d u c t i o n of F e ( I I I ) to F e ( I I ) . o Since F e ( I I ) has a l a r g e r i o n i c r a d i u s , 0.74A compared w i t h that of o F e ( I I I ) , 0.64A, F e ( I I ) was more r e a d i l y d i s s o c i a t e d from the porphyrin -119-macrocycle, thus protoporphyrin IX was obtained. The f i n a l product meso-protoporphyrin IX was obtained by c a t a l y t i c hydrogenation of the v i n y l groups of protoporphyrin IX. F e r r i - p r o t o p o r p h y r i n IX (2.0g) was suspended i n formic a c i d (90%; 165 ml) c o n t a i n i n g palladium oxide (460 mg) i n a 250 ml round-bottom f l a s k f i t t e d w i t h a r e f l u x condenser. The whole arrangement was protected from l i g h t by aluminium f o i l . The mixture was r e f l u x e d f o r 1% h r , and the progress of the r e a c t i o n was followed s p e c t r o s c o p i c a l l y . This was achieved by withdrawing an a l i q u o t of the r e a c t i o n mixture, and then d i l u t i n g w i t h formic a c i d . The v i s i b l e a bsorption spectrum was then compared w i t h that of protoporphyrin IX (Figures 8.1 and 8.2). Completion of the r e a c t i o n was determined by the disappearance of the prot o p h y r i n IX spectrum. The palladium oxide was f i l t e r e d o f f using a s i n t e r e d g l a s s funnel of f i n e p o r o s i t y , and was washed w i t h formic a c i d The f i l t r a t e was then poured i n t o 30% ammonium acetate s o l u t i o n (600 ml) w i t h e f f i c i e n t s t i r r i n g . A f t e r being allowed to stand f o r 45 min, the p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and washed repeatedly w i t h deionised water. The p r e c i p i t a t e was taken up i n 2% aqueous ammonia s o l u t i o n (140 ml) to give a brownish-red s o l u t i o n . The sodium s a l t of meso-protoporphyrin IX was " s a l t e d out" w i t h 30% sodium t a r t r a t e s o l u t i o n (24 ml). The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , and i f the supernatant remained brownish-red, the above procedure was repeated. The sodium s a l t was converted i n t o meso-protoporphyrin IX hydro-c h l o r i d e by the a d d i t i o n of b o i l i n g 25% h y d r o c h l o r i c a c i d (120 ml) to the p r e c i p i t a t e i n the c e n t r i f u g e tubes. The s o l i d d i s s o l v e d i n s t a n t l y and c r y s t a l s of the h y d r o c h l o r i d e formed almost immediately. The -120-c r y s t a l l i n e meso-protoporphyrin IX h y d r o c h l o r i d e was c o l l e c t e d by f i l t r a t i o n , washed w i t h a l i t t l e 2.5% h y d r o c h l o r i c a c i d s o l u t i o n , and d r i e d i n a i r . Further d r y i n g was achieved i n a d e s i c c a t o r con-t a i n i n g p e l l e t s of sodium hydroxide. - 1 2 1 -_ J I I I I I L 349 U2U 500 551 625 700 W a v e l e n g t h n m F i g u r e 8 . 1 . V i s i b l e a b s o r p t i o n s p e c t r u m o f p r o t o p o r p h y r i n I X i n f o r m i c a c i d . -122-I I 1 I I I L 306 383 460 473 548 624 700 WAVELENGTH nm F i g u r e 8.2. V i s i b l e a b s o r p t i o n spectrum o f meso-protoporphyrin IX i n f o r m i c a c i d . -123-8.2 P r e p a r a t i o n of Non-Radioactive Indium-Mesoprotoporphyrin IX M a t e r i a l s : Indium c h l o r i d e , . I n C l ^ (anhydrous, u l t r a - p u r e ) was obtained from A l f a ; reagent g l a c i a l a c e t i c a c i d from A l l i e d Chemical Canada, L t d . ; s i l i c a g e l (70-140 mesh) f o r column chromatography was obtained from Macherey, Nagel & Co., Germany; and meso-protoporphyrin IX was prepared i n the l a b o r a t o r y according to the procedure i n Section 8.1. Procedure: In a 250 ml one-necked round-bottom f l a s k f i t t e d w i t h a r e f l u x condenser, indium c h l o r i d e (0.148g) and meso-protoporphyrin IX (0.200g) were heated to b o i l i n g i n g l a c i a l a c e t i c a c i d (160 ml) c o n t a i n i n g sodium acetate (0.717g). The r e a c t i o n was protected from l i g h t by aluminium f o i l . R e f l u x i n g was continued f o r 4% hr. Completion of the m e t a l l a t i o n process was determined s p e c t r o s c o p i c a l l y by the disappearance of the v i s i b l e spectrum of meso-protoporphyrin IX. The s o l u t i o n was con-centrated on a r o t a r y evaporator to a minimal volume. A d d i t i o n of water to the warm, concentrated s o l u t i o n produced a copious p r e c i p i t a t e of b r i g h t - r e d indium-mesoprotoporphyrin IX (InMPP I X ) . The s o l i d was c o l l e c t e d , washed w i t h water and d r i e d over p e l l e t s of sodium hydroxide i n vacuo. -124-8.2a Thin-layer chromatography A n a l y t i c a l thin-layer chromatography ( t i c ) was used to check the p u r i t y of InMPP IX. Eastman chromatogram sheets of s i l i c a gel with fluorescent indi c a t o r were used. After an extensive search, the best solvent system for the development of the chromatograms was a mixture of toluene/methanol (2:1). No spraying agent was required to see the spots since components of i n t e r e s t were intensely colored. Figure 8.3 shows the t i c r e s u l t s with various solvent systems. A B T : M ( 1 : 1 ) O R I G I N T:M(2:1) DH(5--1) T : M(10 - - 2 ) Figure 8.3. TLC of (A) meso-tetraphenylporphine IX and (B) indium meso-tetraphenylporphyrin IX Abbreviations: solvents: T, toluene; M, methanol; H, water; L, 2,6-Lutidine. -125-8.2b P u r i f i c a t i o n of Indium-Mesoprotoporphyrin IX From the t i c r e s u l t s (Section 8.2a), the crude InMPP IX complex (100 mg) was d i s s o l v e d i n minimal methanol and chromatographed on a 62 cm x 1.6 cm column of s i l i c a g e l packed i n toluene. The column was e l u t e d w i t h toluene/methanol (2:1). InMPP IX was eluted f i r s t followed by unreacted MPPIX. A l l f r a c t i o n s were checked s p e c t r o s c o p i c a l l y and by t i c . The solvent was removed i n vacuo to recover the b r i g h t - r e d InMPPIX. 8.2c V i s i b l e Absorption Spectroscopy The v i s i b l e a bsorption spectra of indium-mesoprotoporphyrin IX i n formic a c i d and benzene were recorded on a Cary Model 14 spectro-photometer. On formation of InMPP, the i n t e n s i t y of the ct-band increased s i g n i f i c a n t l y to almost that of the 8-band (see Figures 8.4 and 8.5). The two bands were hypochromically s h i f t e d , the ot-band has A at 573 nm and the 8-band at 560 nm. -126-399 5 3 6 5 7 3 I I L _ l I I I 335 460 UU 548 625 Wavelength nm F i g u r e 8.4. V i s i b l e a b s o r p t i o n spectrum of indium meso-protoporphyrin I X i n formic a c i d . - 1 2 7 -F i g u r e 8.5. i n benzene. V i s i b l e a b s o r p t i o n spectrum of indium m e s o - p r o t o p o r p h y r i n IX - 1 2 8 -8.3 P r e p a r a t i o n of Radioactive I n d i u m - I l l Meso-protoporphyrin IX The normal method of preparing indium-MPP i s one of r e f l u x i n g l a r g e excess of indium and the porphyrin as discussed i n S e c t i o n 8.2. This method i s not a p p l i c a b l e to the synth e s i s of r a d i o a c t i v e i n d i u m - I l l MPPIX s i n c e the molar r a t i o of r a d i o a c t i v e i n d i u m - I l l to MPPIX i s extremely low. Therefore a s l i g h t l y d i f f e r e n t method was developed. The search f o r the optimum r e a c t i o n c o n d i t i o n s was c a r r i e d out w i t h " c o l d " indium i n view of the f a c t that i n d i u m - I l l emits gamma rays. Several f a c t o r s were considered: r e f l u x i n g time, amount of " c o l d " indium present was c r i t i c a l s i n c e both "hot" and " c o l d " indium would compete f o r the porphyrin, and the amount of MPP. The method described here was obtained a f t e r an extensive search f o l l o w i n g sometimes d i s -a p p o i n t i n g r e s u l t s of low r a d i o a c t i v i t y of the r e c o n s t i t u t e d myoglobin w i t h ^^InMPPIX. A high r a d i o a c t i v i t y of ^ ''"InMPPIX was e s s e n t i a l to the success of the subsequent gamma-gamma experiments on the r e c o n s t i t u t e d myoglobin. A l l the experiments were c a r r i e d out w i t h i n an improvized lead (3 mm t h i c k ) enclosure of 50 cm x 50 cm x 40 cm i n a fume-hood. This placed a severe l i m i t a t i o n on the v i s i b i l i t y and chemical manipu-l a t i o n s .Since the h a l f - l i f e of ind ium-111 i s short (tj=2.8 days), a l l glasswares and r a d i o a c t i v e contaminated s o l u t i o n s and a r t i c l e s were stored i n the lead enclosure u n t i l the r a d i o a c t i v i t y was no longer d e t e c t a b l e before d i s p o s a l . A P i c k e r Model 642081 Labmonitor was used f o r determining r a d i a t i o n l e v e l s , e s t i m a t i n g sample a c t i v i t y , and checking contamination. M a t e r i a l s : In-111 was obtained commercially as c a r r i e r - f r e e •'•^^InClo i n -139-aqueous s o l u t i o n c o n t a i n i n g 0.45% to 0.9% sodium c h l o r i d e from Medi-Physics Corp., E m e r y v i l l e , C a l i f o r n i a . Procedure: The method was e s s e n t i a l l y s i m i l a r to that described i n S e c t i o n 8.2. In a 25 ml round-bottom f l a s k f i t t e d w i t h a r e f l u x condenser, MPP IX (6 mg), " c o l d " I n C l 3 (2.3 mg), sodium acetate (9.1 mg) i n g l a c i a l a c e t i c a c i d (6 ml) was s t i r r e d v i g o r o u s l y . ^ " ^ I n C l ^ s o l u t i o n (1.1 ml; 2.2 mCi) was i n j e c t e d i n t o the r e a c t i o n mixture which was r e f l u x e d f o r 6-7 hr. The s o l u t i o n was then concentrated to a minimal volume by evaporation. A d d i t i o n of c o l d deionized water to the warm, concentrated s o l u t i o n produced a b r i g h t - r e d p r e c i p i t a t e which was washed repeatedly w i t h c o l d water and a i r - d r i e d . No attempt was made to p u r i f y the product. The r a d i o a c t i v i t y of the product was checked w i t h the Labmonitor. The "^"'"InMPPIX was then immediately used f o r the r e c o n s t i t u t i o n experiments (See Section 8.5). •-130-8.4 Pr e p a r a t i o n of Apomyoglobin S o l u t i o n M a t e r i a l s : Myoglobin from spermwhale s k e l e t a l muscle type I I ( s a l t - f r e e l y o p h i l l i s e d powder) was obtained from Sigma. 2-Butanone (methyl e t h y l ketone) was obtained from F i s h e r . Procedure: The procedure was s i m i l a r to that reported by Breslow (151). A 500 mg sample of myoglobin was d i s s o l v e d i n 60 ml deionized water (VL% s o l u t i o n ) at 0°C, and the pH c a r e f u l l y adjusted to 1.5 w i t h IN HC£. The removal of the heme from myoglobin was insured i f the pH of the i n i t i a l met-Hb s o l u t i o n was near 1.5 which i s a c r i t i c a l value f o r the cleavage of the protein-heme l i n k . The s o l u t i o n was then ex t r a c t e d at 4°C f i r s t w i t h an equal volume of 2-butanone, then twice more w i t h a half-volume of 2-butanone to remove the heme. The l a s t e x t r a c t i o n was g e n e r a l l y found to be superfluous. The r e s u l t i n g , s l i g h t l y straw-colored s o l u t i o n was then immediately d i a l y z e d at 4°C against 4 changes of sodium bicarbonate s o l u t i o n (50 mg/l l i t r e ) , and then against s e v e r a l changes of deionized water. The apomyoglobin s o l u t i o n was -4 f u r t h e r d i a l y z e d against 2 changes of 1 x 10 M disodium EDTA s o l u t i o n , and against s e v e r a l changes of deionized water. The apomyoglobin s o l u t i o n was c e n t r i f u g e d to remove denatured p r o t e i n which had p r e c i p i t a t e d , and was concentrated to M.0 ml by u l t r a - f i l t r a t i o n using an Amicron arrangement f i t t e d w i t h a UM-2 membrane (>1000 M.W.). F i n a l l y , the concentrated p r o t e i n s o l u t i o n was d i a l y z e d against 4 changes of 250 ml sodium borate b u f f e r (pH 9.2; i o n i c strength 0.16). The conc e n t r a t i o n of apomyoglobin was determined based upon i t s absorbance at 280 nm (e = 15,500). The apomyoglobin s o l u t i o n was immediately used f o r r e c o n s t i t u t i o n w i t h 1 1 : LInMPPIX (See Secti o n 8.5). -132-8.5 R e c o n s t i t u t i o n of Apomyoglobin w i t h I n d i u m - I l l Meso-protoporphyrin IX A l l the r e c o n s t i t u t i o n experiments were c a r r i e d out w i t h i n the lead enclosure i n a fume-hood as discussed i n S e c t i o n 8.3. As mentioned i n Section 8.3, the l i m i t e d v i s i b i l i t y (due to the lead enclosure) had been a severe handicap, hence a lead glass s h i e l f (32 cm x 24 cm) was used during column chromatography. M a t e r i a l s : Sephadex G-25 was obtained from Pharmacia Fi n e Chemicals, Sweden; carboxy methyl c e l l u l o s e , CM-52 (microgranular, preswollen), a c a t i o n exchanger f o r column chromatography was obtained from Whatman, England. ^"^InMPPIX was obtained from the method discussed i n Sectio n 8.3. Apo-myoglobin was prepared as described i n Sectio n 8.4. Procedure: The procedure of r e c o n s t i t u t i o n was that of S r i v a s t a v a (152). "'""'""''InMPPIX was d i s s o l v e d i n a minimal volume of 0.1N NaOH and immediately d i l u t e d t e n - f o l d w i t h borate b u f f e r (pH=9.2; i o n i c strength = 0.16). The ^''"InMPPIX s o l u t i o n was immediately added to 4 ml of apomyoglobin s o l u t i o n -4 [1.6 x 10 H/l] at 0°C w i t h s t i r r i n g . The r e s u l t i n g s o l u t i o n was then stored at 0°C i n a Dewar f l a s k f o r about 8 hr. Normal r e f r i g e r a t i o n was i m p r a c t i c a l i n view of the high gamma emissions. The s o l u t i o n was then chromatographed over Sephadex G-25 (48 cm x 1 cm column) which had been e q u i l i b r a t e d w i t h 0.005M phosphate b u f f e r , pH6.3 and eluted w i t h the same b u f f e r . This step removed excess unreacted "''"''"'"InMPPIX, A red d i s h band corresponding to the r e c o n s t i t u t e d myoglobin was eluted w i t h i n the vo i d volume of the column followed by the unreacted "'^InMPPIX. To remove "'""'""'"InMPPIX that were bound to non-active s i t e s on the p r o t e i n s , -133-the eluted p r o t e i n was absorbed on a CM-52 c e l l u l o s e c a t i o n exchange column (8 cm x 2 cm) e q u i l i b r a t e d w i t h 0.005M phosphate, pH 6.3 (below the i s o e l e c t r i c p o i n t , since the p l value of myoglobin i s 7.0). The CM-column was constructed from a 30 cc p l a s t i c disposable syringe as shown i n Figure 8.6. The p r o t e i n absorbed s t r o n g l y on the c e l l u l o s e w h i l e the InMPPIX passed s t r a i g h t through. E l u t i o n was continued u n t i l no ^""^InMPPIX (reddish c o l o r ) was c o l l e c t e d . The eluent was f i n a l l y changed to 0.05M phosphate, pH7.0, the p r o t e i n (a deep red band) immediately moved down the column and was c o l l e c t e d . The r a d i o -a c t i v i t y of the r e c o n s t i t u t e d sample was estimated w i t h the P i c k e r Labmonitor. -134-r u b b e r -s e p t u m s y r i n g e n e d d l e g l a s s w o o l s y r i n g e n e d d l e r u b b e r c o r k 3 0 c c p l a s t i c d i s p o s a b l e s y r i n g e C M - 5 2 g e l s a n d T y g o n t u b i n g F i g u r e 8.6. A s c h e m a t i c d r a w i n g o f a CM-52 c o l u m n c o n s t r u c t e d f r o m a 3 0 c c p l a s t i c d i s p o s a b l e s y r i n g e . - 1 3 5 -8.6 Perturbed Angular C o r r e l a t i o n Measurements The experimental technique i s concerned w i t h the measurement of the angular d i s t r i b u t i o n of the second gamma emission w i t h respect to the d i r e c t i o n of the f i r s t gamma emission. This r e q u i r e s detectors and e l e c t r o n i c equipment which w i l l be described b r i e f l y below. 8.6a Detectors S c i n t i l l a t i o n detectors instead of Geiger counters were used because of t h e i r high e f f i c i e n c y and good energy r e s o l u t i o n . They a l s o provide f a s t e r pulses w i t h much l e s s u n c e r t a i n t y i n time than Geiger counters which i s e s s e n t i a l i n measurements r e q u i r i n g coincidence d e t e c t i o n . T h a l l i u m - a c t i v a t e d sodium i o d i d e detectors were used f o r the de t e c t i o n of gamma emissions. The high Z of the iod i n e provides a lar g e p h o t o e l e c t r i c c r o s s - s e c t i o n which allows one to choose the gamma-ray d e s i r e d by pulse-height s e l e c t i o n . The r e l a t i v e l y long phosphor decay time of 0.25 ysec may appear to be too long f o r f a s t coincidence work; however, by operating a f a s t coincidence c i r c u i t on the i n i t i a l part of the puls e , r e s o l v i n g times of one-tenth to one-hundred of the decay time could be achieved f o r gamma rays of 100 keV or more. P h o t o m u l t i p l i e r tubes were used to detect pulses from the d e t e c t o r s , and at the same time to provide an a m p l i f i c a t i o n f a c t o r up to 10 . The a m p l i f i e d e l e c t r i c a l pulses were s u f f i c i e n t l y l a r ge to operate the e l e c t r o n i c d e t e c t i o n system. The p h o t o m u l t i p l i e r s were protected from high magnetic f i e l d w i t h magnetic s h i e l d i n g . -136-8.6b E l e c t r o n i c s A s i m p l i f i e d schematic diagram of the f a s t - s l o w coincidence c i r c u i t i s shown i n Figure 8.7. Radiations f a l l i n g w i t h i n the s o l i d angles of the detectors were counted. However, the coincidence analyzer was used to s e l e c t p r i n c i p a l l y only p a i r s of r a d i a t i o n s and which are g e n e t i c a l l y r e l a t e d to each other ( i . e . emitted i n the same nuclear decay). The e l e c t r i c a l pulses from the detectors were chanelled i n t o two l i n e s , " f a s t " and "slow" (slow was i n the order -6 -7 -9 of 10 sec, f a s t was 10 to 10 sec pulse w i d t h ) . A f a s t coincidence c i r c u i t was e s s e n t i a l to d i s c r i m i n a t e against chance coincidences at high counting r a t e s . Pulses on the f a s t l i n e s were a p p l i e d to the f a s t coincidence c i r c u i t . The pulses were at the same time a p p l i e d i n sequence to slow l i n e a r a m p l i f i e r s and to pulse-height analyzers f o r energy s e l e c t i o n . The pulse height was p r o p o r t i o n a l to the energy of the i n c i d e n t photon. The outputs of the analyzers together w i t h the output of the f a s t coincidence c i r c u i t were then a p p l i e d to a slow m u l t i p l e coincidence c i r c u i t that s e l e c t s the event. The output pulse was then passed to a multi-channel analyzer f o r data storage. In the present study, the angular d i s t r i b u t i o n was studied as a f u n c t i o n of the time between the formation of the o r i e n t e d s t a t e and i t s decay, that i s the time between the a r r i v a l of the two gamma rays. This was achieved by i n t r o d u c i n g an e l e c t r o n i c delay i n the l i n e of the f i r s t d e t e c t o r . • 1 3 7 -Amplif ier | Post —\ Amplifier Post Coincidence Circuit Fast Amplifie--J Slow Amplifier Smgie Channel Pulse Height Analyzer Slow Coincidence Cucutl Output Single Chonnel Pulse Height Analyier F i g u r e 8 . 7 . A s c h e m a t i c d i a g r a m f o r a f a s t - s l o w c o i n c i d e n c e a r r a n g e m e n t f o r P . A . C . m e a s u r e m e n t s . From F r a u e n f e l d e r a n d S t e f f e n ( r e f . 1 3 0 ) . -138H 8.6c Method A three-detector f a s t - s l o w gamma ray coincidence spectrometer was used. The detectors were arranged at c a r d i n a l p o i n t s around the r a d i o a c t i v e sample. The P.A.C. measurements at 180° and 90° were performed simultaneously using three 2" x 2" d i a . NaI(T£) d e t e c t o r s . A s i m p l i f i e d diagram of the f a s t - s l o w coincidence c i r c u i t used f o r the present measurements i s shown i n Figure 8.8, i n d i c a t i n g how the angular c o r r e l a t i o n s W(Tr,t) and W(7r/2,t) are routed to separate halves of the multichannel analyzer memory. The y-ray energies were s e l e c t e d i n the slow s i d e of the c i r c u i t by a timing s i n g l e channel analyzer (TSCA). The time-to-amplitude converter (TAC) measured the time i n t e r v a l between the a r r i v a l of the s t a r t (173 keV) and stop (247 keV) pulses, w h i l e the slow coincidence l o g i c determined between which p a i r of detectors the event o r i g i n a t e d . The events were then routed to separate halves of the multichannel analyzer memory. The multichannel analyzer would then provide a d i r e c t d i s p l a y of emission a n i s t r o p y against delay time. The prompt time r e s o l u t i o n of t h i s system was 1.1 ns f o r ^ C o gamma rays. However, w i t h the energy windows set a p p r o p r i a t e l y f o r the 173 keV and 247 keV gamma rays i n "'"^ C^d, t h i s d e t e r i o r a t e d to 2.7 ns. By measuring W(iT,t) and W(ir/2,t) simultaneously, no c o r r e c t i o n f o r the decay of the source was necessary, and improved counting s t a t i s t i c s r e s u l t e d . The s c a l e r counts i n the slow side-channels a u t o m a t i c a l l y provide the required n o r m a l i z a t i o n s . Gain s t a b i l i z e r s ensured long-term s t a b i l i t y i n the l i n e a r pulses from the a m p l i f i e r s and an t i - c o i n c i d e n c e requirements were used to e l i m i n a t e " c r o s s - t a l k " i n the counting system. n 90' SLOW 1FAST LA T.S.C.A Y 2 |SCALER \-0° SLOWr FAST — r X L 180° SLOW FAST LA FASTI OR T.S.C.A SCALER 1 SLOW COIN ANTI T.S.C.A v 2 STARTI tSTOP T. A . C •HSCALER co SLOW COIN -4v 1 LIN. LIN. GATE GATE MIXER MIXER MCA 90° MCA 180° F i g u r e 8.8. A s i m p l i f i e d b l o c k d i a g r a m o f t h e e l e c t r o n i c s u s e d i n t h e p r e s e n t t i m e - d i f f e r e n t i a l p e r t u r b e d a n g u l a r c o r r e l a t i o n e x p e r i m e n t s . ----140-The samples i n s o l u t i o n or s o l i d form were contained i n normal glass n.m.r. tubes and kept at 10-15°C by a stream of c o l d a i r . The samples were adjusted to give a d i s i n t e g r a t i o n r a t e u s u a l l y of the 4 4 order of 4x10 - 6x10 counts per second i n order to reduce the chance coincidence c o n t r i b u t i o n to the true coincidence r a t e . A t y p i c a l t i m e - d i f f e r e n t i a l perturbed angular c o r r e l a t i o n measurement takes about 2 days. The data were corrected f o r s o l i d angle e f f e c t s and f i n i t e time r e s o l u t i o n . The d i f f e r e n t i a l n o n - l i n e a r i t y of the time spectrum was measured w i t h random stop and s t a r t pulses i n the TAC and found to be e s s e n t i a l l y over 90% of the time range. V i s c o s i t y measurements were performed on the p r o t e i n samples using a Ostwald viscometer. The change i n s o l u t i o n v i s c o s i t y was e f f e c t e d by adding predetermined amount of g l y c e r i n e . The s o l i d form of p r o t e i n was obtained by l y o p h i l i z a t i o n of the p r o t e i n s o l u t i o n . -141-CHAPTER IX RESULTS AND DISCUSSION 9.1 RESULTS-Time-Differential Perturbed Angular C o r r e l a t i o n s Data The r e c o n s t i t u t e d ^^InMPP-Mb was studied i n 0.05M sodium phosphate b u f f e r , pH 7.0, and i n l y o p h i l i z e d powder form at 12°C. The v i s c o s i t y of the aqueous sample was changed by adding g l y c e r i n e . In the samples f o r the t i m e - d i f f e r e n t i a l perturbed angular c o r r e l a t i o n (TDPAC) e x p e r i -ments, no attempt was made to ensure complete i n c o r p o r a t i o n of ^"^InMPP IX i n t o apomyoglobin. The excess apomyoglobin should pose no d i f f i c u l t y s i n c e only those myoglobin molecules c o n t a i n i n g ^^"InMPP IX w i l l con-t r i b u t e to the TDPAC s i g n a l . The TDPAC spectra f o r 1 1 1InMPP-Mb i n aqueous b u f f e r w i t h and without g l y c e r i n , and i n l y o p h i l i s e d powder form are shown i n Figures 9.1-9.3. Results f o r the f r e e 1 : L 1InMPP IX i n dimethyl formamide (DMF) and 1 : L 1 I n T P P : C l i n chloroform (CHCl^) are i l l u s t r a t e d i n Figures 9.4-9.5. The s o l i d curves are l east-square f i t s to the experimental data. The p e r t u r b a t i o n f a c t o r s ^ 2 2 ( t ) w e r e determined from the measured W(iT,t) and W ( T r/2,t) r (t\ _ _2 W(TT,t)-W(Tt/2,t) (6.5) 22 v ' A 2 W(TT,t)+2W(ff/2,t) where A 2 = -0.18. The data were correct e d f o r s o l i d angle e f f e c t s w i t h AC0R " W V W where Q are the s o l i d angle c o r r e c t i o n f a c t o r s . (9.1) For the aqueous samples, a t h e o r e t i c a l f i t to the measured p e r t u r b a t i o n f a c t o r s was made using the f o l l o w i n g f u n c t i o n G 2 2 ( t ) = (1-A) + Aexp( t / x c ) G 2 ( t ) (9.2) where G 2 ( t ) i s the s t a t i c r e s u l t of p o l y c r y s t a l l i n e powder. For a non-a x i a l l y symmetric e l e c t r i c g r a d i e n t , represented by the asymmetry, n and a f i n i t e spread, <5 i n the quadrupole i n t e r a c t i o n , G 2 ( t ) i s given by 3 G„(t,n,6) = E b (n)exp(-%w 2T 2)exp(-J2co 2 6 2 t 2 ) COS(OJ t ) (9.3) z n n c n n n=o 2 The f a c t o r exv>(-h(ii T ) allows f o r f i n i t e time r e s o l u t i o n and the n c quadrupole frequencies are given as u (n) = f (n)u (9.4) n n o The f ( n) values are obtained from the d i a g o n a l i z a t i o n of the non-symmetric f i e l d Hamiltonia (141). For n=o ( a x i a l symmetry) f ( l ) take on the i n t e g r a l values 1, 2, and 3. For nfo, the w n ( n ) a r e no longer harmonics. 2 The program MINUIT (153) was used to minimize the value of x i n the space of f r e e parameters, TC, OJq, and 6 (the r e l a t i v e width parameter). For the s o l i d sample where there was no motion at a l l (T -*»), the f o l l o w i n g f u n c t i o n was used to f i t the TDPAC data G 2 2 ( t ) = G s ( t , n , 6 ) (9.5) -143-The f i t t e d values obtained f o r T , r\, and 6 f o r each of the case are compiled i n Table 9.1 Table 9.1. Parameters derived from a n a l y s i s of TDPAC spectra f o r llllnMPP-Mb s o l i d and i n aqueous b u f f e r s o l u t i o n . A T (ns) c 0) (MHz) o n 6 L y o p h i l i z e d ( s o l i d ) p r o t e i n 1.0 00 36±4 0.01±0. 01 0. 37±0. 03 Aqueous p r o t e i n c o n t a i n i n g 41% g l y c e r i n e 0.98±0.01 30±3 0.01±0. 01 0. 36±0. 04 Aqueous p r o t e i n without g l y c e r i n e 0.96±0.01 « 3 25±3 0.00±0. 01 0. 42±0. 04 09 D I 1 1 1 1 1 1 1 1 0.0 18.125 36.25 54.375 72.5 90.625 108.75 126.88 145.0 CHANNEL NUMBER F i g u r e 9 . 1 . T i m e - d i f f e r e n t i a l p e r t u r b e d a n g u l a r c o r r e l a t i o n s p e c t r u m f o r ^ I n M P P - M b i n a q u e o u s s o d i u m p h o s p h a t e b u f f e r (pH = 7 . 0 ) w i t h g l y c e r i n e a d d e d t o g i v e a f i n a l g l y c e r i n e p e r c e n t a g e o f 4 1 . 0 . 6 9 n s p e r c h a n n e l . 1 1 1 1 1 1 1 1 1 0 . 0 18.125 3 6 . 2 5 5 4 . 3 7 5 7 2 . 5 9 0 . 6 2 5 108.75 126.88 CHANNEL NUMBER F i g u r e 9 . 2 . T i m e - d i f f e r e n t i a l p e r t u r b e d a n g u l a r c o r r e l a t i o n s p e c t r u m f o r ^ I n M P P - M b i n a q u e o u s s o d i u m p h o p s h a t e b u f f e r (pH = 7 . 0 ) w i t h o u t g l y c e r i n e . 0 . 6 9 n s p e r c h a n n e l . cn 0.0 1 1 1 1 1 18.125 36.25 54.375 72.5 90.625 108.75 126.88 CHANNEL NUMBER F i g u r e 9 . 3 . T i m e - d i f f e r e n t i a l p e r t u r b e d a n g u l a r c o r r e l a t i o n s p e c t r u m f o r ^ ^ I n M P P - M b i n t h e l y o p h i l i z e d powder f o r m . 0 . 6 9 n s p e r c h a n n e l . 145.0 F i g u r e 9.4. T i m e - d i f f e r e n t i a l p e r t u r b e d a n g u l a r c o r r e l a t i o n s p e c t r u m f o r ] 1 1 I n M P P IX i n d i m e t h y l f o r m a m i d e . lO »8.i25 3B~25 54.375 72.5 90.625 108.75 126.88 145 D CHANNEL NUMBER F i g u r e 9.5. T i m e - d i f f e r e n t i a l p e r t u r b e d a n g u l a r c o r r e l a t i o n s p e c t r u m f o r ^ I n T P P i n c h l o r o f o r m . - 1 4 9 -9.2 DISCUSSION 9.2a A n a l y s i s of the T l m e - D l f f e r e n t l a l Perturbed Angular C o r r e l a t i o n Spectra 111 The q u a l i t a t i v e appearance of the l y o p h i l i z e d InMPP-Mb TDPAC data (Figure 9.3) confirms that "'"''"''"InMPP IX i s t i g h t l y bound to myoglobin and i s experiencing a s t a t i c quadrupolar i n t e r a c t i o n . The experimental curve does not show the normal c h a r a c t e r i s t i c p e r i o d i c behavior of the d i f f e r e n t i a l G22^^ ^ o r s t a t i c i n t e r a c t i o n s as o c c u r r i n g i n p o l y c r y s t a l l i n e samples (see Figure 6.6 i n Se c t i o n 6.2b). Instead the l y o p h i l i z e d p r o t e i n sample shows f i r s t a r a p i d decay and then f l u c t u a t e s around the low value . This non-p e r i o d i c observation i s probably due to e f f e c t s a r i s i n g from the e x c i t e d e l e c t r o n s h e l l a f t e r K-capture during the t r a n s i t i o n from "'""'""'"In to the e x c i t e d "'""'"''"Cd s t a t e (see Appendix C) . Lehmann and M i l l e r a l s o observed s i m i l a r behavior f o r the two non-metallic sources In^O^ and In(OH)^, as discussed i n S e c t i o n 6.2b. Figure 9.2 gives the TDPAC r e s u l t s f o r ''"''""'"InMPP-Mb i n aqueous b u f f e r s o l u t i o n without g l y c e r i n e at 12°C. The angular c o r r e l a t i o n i s s t r o n g l y perturbed. This i n d i c a t e s that "'""'""'"InMPP IX i s bound and i s i n a r e l a t i v e l y immobile environment ( i . e . , long r o t a t i o n a l c o r r e l a t i o n time). As a c o n t r o l , TDPAC experiments were performed on f r e e "'""'""'"InMPP IX ( i . e . , i n the absence of p r o t e i n ) i n DMF and 1 1 : L I n T P P : C l i n CHC1 3. The r e s u l t s are shown i n Figures 9.4 and 9.5. In contrast to the TDPAC r e s u l t s obtained f o r the "'""'""''InMPP-Mb i n aqueous b u f f e r , the angular c o r r e l a t i o n i s e s s e n t i a l l y unperturbed or i s o t r o p i c f o r the f r e e '''''""''InMPP IX and "'""'"^InTPPrCl. This observation i m p l i e s much f a s t e r molecular r o t a t i o n f o r the l a t t e r case which leads to the much slower exponential decay of anisotropy. -150-The attentuation factors G 2 2 ( t ) observed for the above sol u t i o n samples can be explained i n terms of the influence of molecular r o t a t i o n i n s o l u t i o n on anisotropy. As discussed i n Section 6.2a i n Chapter VI, Abragam and Pound showed that for small molecules or ions randomly oriented i n d i l u t e solutions the d i f f e r e n t i a l G 2 2 ( t ) takes the form of a simple exponential decay G 2 2 ( t ) = exp(-A 2/t) (6.6) where X2 = T 0 0 ( e 2 q Q ) 2 x c / h 2 <6-7> It i s clear from Eq. 6.7 that A 2 i s d i r e c t l y proportional to r o t a t i o n a l c o r r e l a t i o n time, x which i s usually small for small c molecules; T =10 "'""''sec. Consequently, from Eqs. 6.6 and 6.7, a short x^ w i l l give r i s e to a small attentuation of the angular c o r r e l a t i o n s i n s o l u t i o n . This explains why the anisotropy of angular c o r r e l a t i o n s remains l a r g e l y unperturbed for small molecules such as "'""''"'"InMPP IX and "'""'""'"InTPPrCl. However, for large macromolecules l i k e myoglobin (MW = 17,000), x^ as expected i s large, which accounts for the low G 2 2 ( t ) value and the strongly perturbed angular c o r r e l a t i o n for the aqueous protein sample. The form of G 2 2 ( t ) for the aqueous protein sample without glycerine i s s i m i l a r to that obtained i n the l y o p h i l i z e d protein sample. This suggests a q u a s i s t a t i c i n t e r a c t i o n i n which G 2 2 ( t ) i s i t s e l f time-dependent. The t h e o r e t i c a l f i t to the data shows that G 2 2 ( t ) f a l l s to a c h a r a c t e r i s t i c minimum followed by a secondary maximum. The l a t t e r i s smeared out, presumably by inhomogeneities in the V^ z (component of the e l e c t r i c f i e l d gradient i n the z - d i r e c t i o n ) or from the a f t e r -e f f e c t s of e l e c t r o n capture, which wipe out the angular c o r r e l a t i o n s . A s l i g h t l y more s i g n i f i c a n t a t t e n t u a t i o n i s observed i n the s o l i d case than i n aqueous sample. However, the c l o s e r e l a t i o n s h i p between the s o l i d and aqueous p r o t e i n samples i s c o n s i s t e n t w i t h the idea that ''"''"'''InMPP IX i s incorporated i n t o the a c t i v e - s i t e on the apomyoglobin. As discussed i n Secti o n 6.2a, the 3-dimensional molecular r o t a t i o n a l c o r r e l a t i o n time i s given by the equation below 4 R 3 T c = 3 K T - 5 ( 6 ' 1 0 ) I t can be seen from Eq. 6.10 that the d i r e c t r e l a t i o n s h i p between and v i s c o s i t y (?) suggests that changing the v i s c o s i t y of the medium would cause a corresponding change i n anisotropy of the angular c o r r e l a t i o n s . In the aqueous p r o t e i n sample c o n t a i n i n g 41% g l y c e r i n e , the increase i n s o l u t i o n v i s c o s i t y leads to a longer r o t a t i o n a l c o r r e l a t i o n time f o r the p r o t e i n . This slower molecular motion (long T ) causes a r a p i d decrease i n the anisotropy (Figure 9.1), i n accordance w i t h Eqs. 6.6 and 6.7. The more pronounced G^it) minimum suggests a c l o s e r s i m i l a r i t y between the l y o p h i l i z e d p r o t e i n and the aqueous p r o t e i n sample c o n t a i n i n g 41% g l y c e r i n e than the one w i t h no g l y c e r i n e . 9.2b Lease-Squares Analyses of the T i m e - D i f f e r e n t i a l Perturbed Angular  C o r r e l a t i o n Data The l e a s t - s q u a r e s analyses of the TDPAC data using v a r i o u s f u n c t i o n s f o r the t i m e - d i f f e r e n t i a l (^(O i n c l u d e (a) a pure s t a t i c i n t e r a c t i o n which corresponds to Eq. 9.5, and (b) a s i n g l e s t a t i c i n t e r a c t i o n combined w i t h r o t a t i o n a l d i f f u s i o n ( i . e . , m u l t i p l i c a t i v e combination of a s t a t i c and time-dependent i n t e r a c t i o n s ) as expressed by Eq. 9.2. I t can be seen from Figures 9.1-9.3 that the t h e o r e t i c a l f i t s are adequate f o r the e a r l y TDPAC data (<40 nsec) but are r a t h e r poor f o r l a t e r times (>40 nsec) Therefore, f o r both aqueous and s o l i d measurements, the e a r l y TDPAC data should be more r e l i a b l e and meaningful. In a d d i t i o n e a r l y TDPAC data has a greater s t a t i s t i c a l accuracy and i n t r i n s i c a l l y contains more s i g n i f i c a n t i n f o r m a t i o n concerning quadrupolar i n t e r a c t i o n s , whereas the l a t e r data i s more prone to experimental e r r o r s . The s o l i d curve i n Figure 9.3 represents the t h e o r e t i c a l f i t c a l c u l a t e d using Eq. 9.5 f o r the l y o p h i l i z e d p r o t e i n TDPAC data. The curve i s c h a r a c t e r i s t i c of a s i n g l e s t a t i c quadrupolar i n t e r -a c t i o n . Agreement w i t h the e a r l y experimental data i s good. However, the r e q u i r e d value of 6 ( d i s t r i b u t i o n i n quadrupole frequency) i n the f i t was s u f f i c i e n t l y l a r g e to wipe out a l l the f i n e s t r u c t u r e s at l a t e r times as r e f l e c t e d by the poor agreement w i t h the l a t e r TDPAC data. The f i t t e d asymmetry parameter, n=0.01±0.01 i s c o n s i s t e n t w i t h the expectation that the e l e c t r i c f i e l d gradient around "^^"InMPP IX 111 should be a x i a l l y symmetric i n the square-pyramidal IriMPP IX complex. The TDPAC data f o r both the aqueous samples (with and without g l y c e r i n e ) were i n t e r p r e t e d on the b a s i s of a combination of a s t a t i c and a time-dependent i n t e r a c t i o n mechanism. The s o l i d curves i n Figures 9.1 and 9.2 represent the t h e o r e t i c a l curves c a l c u l a t e d using Eq. 9.2 f o r the two aqueous samples. The good agreement w i t h the e a r l y TDPAC data, together w i t h the f a c t that the f i t t e d n=o, show that the "^^InMPP IX i s indeed experiencing a s t a t i c i n t e r a c t i o n due to i t s -153-surrounding e l e c t r o n s , as w e l l as a time-dependent i n t e r a c t i o n a r i s i n g from the r o t a t i o n a l d i f f u s i o n of the p r o t e i n molecule. The s i t u a t i o n s f o r both the aqueous samples are s i m i l a r to the r e s u l t s derived by M a r s h a l l and Meares (13) f o r slow molecular motion (see Se c t i o n 6.2c). As i n the case of the l y o p h i l i z e d p r o t e i n , the f i t s are damped out to give an e s s e n t i a l l y f l a t l i n e s at l a t e r times (>40 nsec), again due to the l a r g e r e q u i r e d values of <5. The curves, however, are able to reproduce the e a r l y TDPAC data (<40 nsec) a c c u r a t e l y . The r e s u l t s of the least-squares analyses compiled i n Table 9.1 show that the value of 0)^(3013 MHz) f o r the aqueous p r o t e i n c o n t a i n i n g 41% g l y c e r i n e i s comparable to that of the l y o p h i l i z e d p r o t e i n ( w =36±4 MHz). This c l o s e r e l a t i o n s h i p between these two u> values o o together w i t h the f a c t that the f i t t e d asymmetry parameter,n, i s zero, confirms the correctness of the TDPAC data r e d u c t i o n . 9.2c Comments on the R o t a t i o n a l C o r r e l a t i o n Times of myoglobin  determined by the Perturbed Angular C o r r e l a t i o n Technique As mentioned e a r l i e r , the 3-dimensional r o t a t i o n a l c o r r e l a t i o n time ( T c) of a s p h e r i c a l molecule i n a l i q u i d medium can be obtained from the f o l l o w i n g equation 3 4^R r , c i r \ \ T c = IRT" 5 ( 6 - 1 0 ) Equation 6.10 can then be used i n two d i f f e r e n t ways to compute T . The f i r s t method r e q u i r e s the value of the Stokes (or hydrodynamic) rad i u s (R), given by the formula below. -154— R = ( 9' 6> rot where K = Boltzman constant, 1.38 x 10 "^erg K ^ T = absolute temperature n = asymmetry parameter D = r o t a t i o n a l d i f f u s i o n c o e f f i c i e n t , determined by rot J sedimentation experiments The Stokes radius (R) for a number of b i o l o g i c a l macromolecules (apomyoglobin, chymotrypsin, hemoglobins) were calculated using published D values according to Eq. 9.6. These Stokes radius values rot were then substituted into Eq. 6.10 to obtain the corresponding values. The r e s u l t s are l i s t e d i n Table 9.2. The second method involves the assumption that protein molecules are r i g i d unhydrated spheres, and each has a p a r t i a l s p e c i f i c volume 3 of 0.73 cm /g. Equation 6.10 can then be rewritten to give Eq. 9.7 as shown below 3 T c = 3~ KT ( 6 - 1 0 ) = (volume/no. of molecules) = (volume/gram)(gram/mole) g (molecules/mole) KT = vM N KT o (9.7) in which M = molecular weight of protein v = partial sepcific volume, 0.73 cm~Vg 3, - J 5 5 — 4 R 3 = volume of a s p h e r i c a l molecule Kl 23 N = Avogadro's number, 6.023 x 10 molecule/mole o £ = v i s c o s i t y of water; 0.01005 g cm ^ sec (c.g.s.) at 20°C; 0.012362 g cm sec at 12°C; 0.008937 g cm at 25°C. The values of T f o r the same b i o l o g i c a l macromolecules were c determined using Eq. 9.7 and are a l s o shown i n Table 9.2. A comparison of these two sets of values i n Table 9.2 shows that the 3-dimensional r o t a t i o n a l c o r r e l a t i o n times c a l c u l a t e d using Eq. 9.7 are a l l smaller than the corresponding values using Eq. 6.10. This can be r e a d i l y explained by reviewing the assumptions made i n the d e r i v a t i o n s of those two equations. In the case of Eq. 9.7, p r o t e i n molecules are assumed to be r i g i d unhydrated spheres w i t h a f i x e d p a r t i a l s p e c i f i c volume. This assumption i s r e a l l y q u i t e a departure from the a c t u a l s i t u a t i o n s i n c e p r o t e i n molecules are known to be e x t e n s i v e l y hydrated. A much b e t t e r approximation i s made i n Eq. 6.10, which i s based on the e f f e c t i v e molecular s i z e of the p r o t e i n from i t s hydrodynamic r a d i u s (R). Hence the higher x^ values computed by the l a t t e r method should be a more accurate r e p r e s e n t a t i o n of the p r o t e i n s ' molecular motions. Table 9.2 a l s o i n c l u d e s some l i t e r a t u r e T values obtained from c ESR and fluorescence d e p o l a r i z a t i o n (F.D) measurements. These r e s u l t s are s i g n i f i c a n t l y d i f f e r e n t from the corresponding x^ _ values computed using the hydrodynamic radius w i t h the Debye equation 6.10. The Table 9.2 Rotational c o r r e l a t i o n times, x and Stokes r a d i i (R), of some proteins Protein Molecular Weight Temperature D „ „ , , C O , 2 0 2 " . (era /s) R(A) from Eq.9.6 T (nsec)from Eq. 9.7 T (nsec)* from Eq.6. T (nsec)from .10° ESR T (nsec)from 3  C F.D. Myoglobin 17,000 25 b 20 11.30x10 12 18.9 tt. 5. 6. .5 .1 .5 6.1 7.0 8.9 8.3 Chymotrypsin 25,000 20 7. .5 12±2f 16.0 Hemoglobin (tertamer) 66,000 20 6.9x10 12 30.9 19. 25. .9 ,2 30.8 38.9 26±28 20.0 Alpha2 (Hb-dimer) 33,000 -7d 20 8.5x10 12 25.1 9. 12. 9 .6 16.5 20.8 12.3 Beta 2 (Hb-dimer) 33,000 20 8.5x10 12 25.1 9. 12. 9 6 16.5 20.8 12.3 a. Adapted from J . Yguerabide (ref. 154). Values were corrected to conform with the usual n.m.r., ESR and the P.A.C. values. b. From Ref. 155. c. From Refs. 156 and 157. d. e From Ref. 158. f From Shimshick et a l . , ( r e f . 159). g From McCallery et a l . , ( r e f . 160) * Assumed R i s constant. - 1 5 1 -disagreement observed may be due to the added e f f e c t of i n t e r n a l r o t a t i o n a l motion of the s p i n - l a b e l or f l u o r e s c e n t - l a b e l , i n a d d i t i o n to the molecular tumbling motion of the whole l a b e l l e d p r o t e i n . In common w i t h other l a b e l l i n g techniques, the P.A.C. method s u f f e r s from the problem of d i s t i n g u i s h i n g l o c a l i s e d motion of probe i t s e l f from the motion of the whole molecule. However, i n the present P.A.C. determination of of myoglobin, t h i s o b s t a c l e was overcome by r e c o n s t i t u t i n g the p r o t e i n . The f e r r o - p r o t o p o r p h y r i n IX (heme) was d i r e c t l y replaced w i t h i n d i u m - I l l mesoprotoporphyrin IX. In t h i s way, the r a d i o i s o t o p e , i n d i u m - I l l was introduced i n t o the a c t i v e s i t e of the p r o t e i n . The X-ray s t r u c t u r e of myoglobin (74, 95) shows that the heme f i t s t i g h t l y i n t o the a c t i v e s i t e . Since ^^InMPP IX i s s t r u c t u r a l l y s i m i l a r to f e r r o - p r o t o p o r p h y r i n IX, there should be very l i t t l e f r e e r o t a t i o n of the ^^InMPP IX at the a c t i v e s i t e of the r e c o n s t i t u t e d myoglobin. Furthermore, the indium-l a b e l l e d p r o t e i n i s very l i k e l y to r e t a i n i t s n a t i v e conformation, i n c ontrast to other l a b e l l i n g techniques i n which there i s always u n c e r t a i n t y as to how much the l a b e l d i s t o r t s the s t r u c t u r e of the p r o t e i n being s t u d i e d . Consequently, s i n c e no a d d i t i o n a l f l e x i b i l i t y i s introduced at the l a b e l attachment s i t e i n the P.A.C. method, the present determination of f o r myoglobin i s probably more accurate than fluorescence d e p o l a r i z a t i o n or ESR determinations. Table 9.3 gives the r o t a t i o n a l c o r r e l a t i o n times (T ) of myoglobin and InMPP IX r e c o n s t i t u t e d myoglobin at v a r i o u s tem-peratures and v i s c o s i t i e s . I t i s important to point out that the r o t a t i o n a l c o r r e l a t i o n times c a l c u l a t e d from Eq. 6.19 at 12°C were based on the Stokes radius (R) of the p r o t e i n at 20°C. These values Rotational correlation times, T c of myoglobin and InMPP reconstituted myoglobin at various temperatures and viscosities (References cited in Table 9.2) Protein Temperature (°C) Viscosity a)(gcm ls'h Tc(nsec)from* Eq. 6.10 Tc(nsec)from P.A.C. TC(nsec)from F.D. Myoglobin 25 20 12 0.0089 0.0100 0.0123 6.1 7.0 8.9 8.3 minMPP-Mb in 0.05M phosphate buffer, pH7.0 without glycerine 12 0.013 9.3 16 (11.5)+ 111InMPP-Mb in 0.05M phosphate buffer, pH7.0 with glycerine to give a final glycerine per-centage of 41% 12 0.049 35.2 22 O * Calculated with R = 18.9A (from Table 9.2) at 20°C, other temperatures and viscosities. and this value is assumed to remain the same at the co Calculated assuming linear relationship between x c and viscosities (from the Debye Equation, Eq. 6.10). -159-should be l a r g e r than i n d i c a t e d i n Table 9.3, si n c e myoglobin i s expected to be more e x t e n s i v e l y hydrated at 12°C and thus have a l a r g e r e f f e c t i v e molecular r a d i u s (R). Res u l t s f o r ^ ^InMPP-Mb i n aqueous b u f f e r without g l y c e r i n e show that the value of T c(P.A.C.) i s greater than that c a l c u l a t e d from the Debye equation (Eq. 6.10) by a f a c t o r of 1.7. This higher experimental i" c(P.A.C.) can be explained by a l l o w i n g f o r a l a r g e r e f f e c t i v e molecular r a d i u s (R) than was used i n the Debye determination. A l t e r n a t e l y , the d i f f e r e n c e s i n may r e f l e c t the f a c t that myoglobin i s not a s p h e r i c a l molecule as i s assumed i n the Debye c a l c u l a t i o n . The d e v i a t i o n from a p e r f e c t sphere causes an increase i n the surface area to volume r a t i o f o r myoglobin, thus i n c r e a s i n g r o t a t i o n a l f r i c t i o n and decreasing the r a t e of molecular tumbling or T . I t i s c l e a r from Table 9.3 that i n order to compare the experimental T c(P.A.C.) value w i t h T C ( f l u o r e s c e n c e ) , i t i s necessary to convert the only a v a i l a b l e (fluorescence) value at 25°C to 12°C. This conversion was done by assuming that v a r i e s l i n e a r l y over a range of v i s c o s i t i e s , as was demonstrated experimentally f o r oxyhemo-g l o b i n by McCallery e t . a l . , (160)(Figure 9.6). The values i n Figure 9.6 are p l o t t e d against the known v i s c o s i t i e s f o r the sucrose concentrations used. Since the v i s c o s i t y of water increases by a f a c t o r of 1.38 on lowering the temperature from 25°C to 12°C, the (fluorescence) of 9.3 nsec at 25°C should be increased by the same f a c t o r to 11.5 nsec at 12°C. The T (fluorescence) at 12°C i s lower than the experimental c T c(P.A.C.) value of 16 nsec. This disagreement may again be due to the i n t e r n a l r o t a t i o n of the f l u o r e s c e n t - l a b e l , i n a d d i t i o n to the molecular motion of the whole p r o t e i n . The present study does however, e f f e c t i v e l y - 1 6 0 -200 r D poise . y( , K )x10J F i g u r e 9 . 6 . R o t a t i o n a l c o r r e l a t i o n t i m e i n n a n o s e c o n d s f o r o x y h e m o g l o b i n as a f u n c t i o n o f n/T i n p o i s e / ° K . Form M c C a l l e r y e t a l . , ( r e f . 1 6 0 ) . -161-demcmstrate that the P.A.C. technique can be used to y i e l d reasonable r o t a t i o n a l c o r r e l a t i o n times f o r p r o t e i n s . The Debye model f o r r e l a x a t i o n (Eq. 6.10) p r e d i c t s that the r o t a t i o n a l c o r r e l a t i o n time should be increased i n the r e c o n s t i t u t e d p r o t e i n s o l u t i o n c o n t a i n i n g 41% g l y c e r i n e r e l a t i v e to that without g l y c e r i n e . According to the d i r e c t l i n e a r r e l a t i o n s h i p between and v i s c o s i t y , one should expect an increase i n the T C(P.A.C.) value of 16 nsec at 0% g l y c e r i n e (5 = 0.013 g cm" 1 s" 1) to 60 nsec at 41% g l y c e r i n e (£ = 0.049 g cm * s * ) , assuming the Stokes ra d i u s (R) remains constant. As i s evident from Table 9.3, the experimental T c(P.A.C.) value at 41% g l y c e r i n e case i s only 22 nsec. However, at high concentrations of g l y c e r i n e the number of f r e e water molecules a v a i l a b l e f o r s o l v a t i n g the p r o t e i n i s reduced. In other words, the h y d r a t i o n sphere i s smaller r e s u l t i n g i n a smaller e f f e c t i v e molecular 3 r a d i u s (R). Since i s p r o p o r t i o n a l to R £, any change i n R w i l l a f f e c t T c more s i g n i f i c a n t l y than an equivalent change i n £. Hence, a decrease i n the hydrated r a d i u s of the r e c o n s t i t u t e d myoglobin ( i n aqueous b u f f e r c o n t a i n i n g 41% g l y c e r i n e ) would cause a more s u b s t a n t i a l r e d u c t i o n i n the T value than can be compensated f o r by the corresponding c increase i n the v i s c o s i t y f a c t o r (£)• Using the Debye equation (Eq. 6.10), the decrease i n the e f f e c t i v e r a d i u s of ^^"InMPP-Mb i n 41% g l y c e r i n e s o l u t i o n can be determined from the experimental T c(P.A.C.) v a l u e s . This c a l c u l a t i o n suggests t h a t , i n 41% g l y c e r i n e s o l u t i o n , the e f f e c t i v e molecular radius f o r myoglobin i s 72% as b i g as i n the absence of g l y c e r i n e (Table 9.4). I t should be s t r e s s e d , however, that Eq. 6.10 represents an o v e r s i m p l i f i c a t i o n , s t r i c t l y a p p l i c a b l e only to i s o t r o p i c motion of -162-s p h e r i c a l molecules. As pointed before, myoglobin i s more of a f l a t t e n e d sphere (74,95), and t h i s f a c t was not taken i n t o account i n the preceding c a l c u l a t i o n s . Table 9.4 Stokes r a d i u s of InMPP-Mb c a l c u l a t e d from the Debye model of r e l a x a t i o n using experimental T (P.A.C.) values P r o t e i n Temp. (°C) V i s c o s i t y ( 5 ) (g c m _ 1 s _ 1 ) T c(P.A.C.) (nsec) Stokes R a d i u s ( R ) + (A) l i : LInMPP-Mb i n 12 phosphate b u f f e r without g l y c e r i n e 0.013 16 22.6 i : L 1InMPP-Mb i n 12 phosphate b u f f e r c o n t a i n i n g 41% g l y c e r i n e 0.049 22 16.2 + Eq. 6.10 was rearranged to give R : 3 |3T kT "\hfr In c o n c l u s i o n , the present study e f f e c t i v e l y demonstrates that the Perturbed Angular C o r r e l a t i o n (P.A.C.) method i s an a t t r a c t i v e , v i a b l e technique f o r o b t a i n i n g r o t a t i o n a l c o r r e l a t i o n times f o r macromolecules. -12 Furthermore, the high s e n s i t i v i t y (as low as 10 M) combined w i t h a b i l i t y f o r in v i v o experimentation makes the P.A.C. method a u s e f u l approach f o r examining other systems of p h y s i o l o g i c a l importance. In a d d i t i o n , i n f o r m a t i o n concerning molecular behavior of p r o t e i n s i n s o l i d and l i q u i d s t a t e s can be obtained. F i n a l l y , a s i m i l a r study has been extended to hemoglobin, i n view of the high l e v e l of i n t e r e s t - 1 6 3 -and c o n t i n u i n g debate on Perutz's stereochemical mechanism f o r oxygen-b i n d i n g c o o p e r a t i v i t y . -164-APPENDIX A CO-OPERATIVE EFFECT OF REVERSIBLE OXYGENATION IN HEMOGLOBIN Hemoglobin (Hb) and myoglobin (Mb) are oxygen c a r r i e r s i n v e r t e b r a t e s . Hemoglobin, which i s contained i n red blood c e l l s , serves as the oxygen c a r r i e r i n blood. On the other hand, myoglobin f a c i l i t a t e s the transport of oxygen i n muscle and a l s o serves as a reserve supply of oxygen i n that t i s s u e . The shape of the oxygen d i s s o c i a t i o n curve of hemoglobin i s sigmoidal whereas that of myoglobin i s h y p e r b o l i c (Figure A . l ) . This curve i s obtained by p l o t t i n g the f r a c t i o n a l s a t u r a t i o n w i t h oxygen versus the p a r t i a l pressure of oxygen gas. However, the chemistry and s t r u c t u r e of myoglobin i s c l o s e l y r e l a t e d to that of the i n d i v i d u a l subunits of hemoglobin. Then as Perutz (6) posed the question,'Why i s i t not good enough f o r the red c e l l to c o n t a i n a simple oxygen c a r r i e r such as myoglobin". The p a r t i a l pressure i n the lungs i s ^100 mm Hg, and as evident from Figures A . l and A.2, both Hb and Mb are almost saturated w i t h oxygen. In the venous blood , as i n the t i s s u e s , much of the oxygen has been released from Hb whereas the myoglobin i s s t i l l s aturated w i t h oxygen. For example at 20 mm Hg, the s a t u r a t i o n of hemoglobin i s about 40% while i n myoglobin i t i s about 80% (see Figure A . l ) . The sigmoid shape of the curve means a greater f r a c t i o n of oxygen i s released more r e a d i l y than the h y p e r b o l i c one. Although Hb and Mb both bind oxygen, but Hb r e l e a s e s i t more r e a d i l y i n order that the t i s s u e s be adequately supplied w i t h oxygen. Myoglobin re l e a s e s oxygen at a much lower p a r t i a l pressure, and i f Mb were the o x y g e n - c a r r i e r , the t i s s u e s would be asphyxiated. Hemoglobin can be d i s s o c i a t e d i n t o i t s c o n s t i t u e n t s u b u n i t s , and -]65-experiments have demonstrated that the i n d i v i d u a l subunits of Hb do not behave d i f f e r e n t l y from myoglobin. The d i f f e r e n c e between Hb and Mb i n oxygen-binding property must then be associated w i t h the i n t e r a c t i o n s between the sub u n i t s , which somehow a l t e r the shape of the e q u i l i b r i u m curve. This i n t e r a c t i o n i s of t e n known as the "heme-heme i n t e r a c t i o n " (126). This expression simply s t a t e s that there are f u n c t i o n a l i n t e r a c t i o n s between heme groups and does not imply d i r e c t i n t e r a c t i o n s or even that the i n t e r a c t i o n s occur between the heme groups i n the same molecule. The sigmoid shape of the ox y g e n - d i s s o c i a t i o n curve of hemoglobin i m p l i e s that the a f f i n i t y of oxygen increases w i t h the increase i n oxygen s a t u r a t i o n which c l e a r l y demonstrates the existence of a co-operative e f f e c t among the four subunits. In other words, the bindi n g of oxygen to the heme group of one subunit has the e f f e c t of i n c r e a s i n g the oxygen a f f i n i t y of the neighbouring subunits of the same molecule. Perutz (6,28,29) proposed a stereochemical i n t e r p r e t a t i o n of the co-operative e f f e c t s i n hemoglobin. For the mechanism of t h i s e f f e c t , o r i g i n a l papers should be consulted. -166-Figure A . l . Oxygen e q u i l i b r i u m curves of A myoglobin and B hemoglobin. -167.-APPENDIX B TETRAPHENYLPORPHINE DIACID Tetraphenylporphine i s v i o l e t i n chloroform but green i n g l a c i a l a c e t i c a c i d . The former c o l o r i s a t t r i b u t e d to the porphyrin f r e e base (Figure B.l) and the l a t t e r to the formation of d i a c i d species (Figure B.2) . The porphinato core of tetraphenylporphine free base e x h i b i t s a marked d e v i a t i o n from p l a n a r i t y of the porphinato core (32,43). S t r u c t u r a l s t u d i e s (32,43) have shown the phenyl groups are almost perpendicular to the mean plane of the porphyrin r i n g because of i n t e r a c t i o n s between the phenyl hydrogens and the outer p y r r o l e hydrogens. This arrangement of the phenyl groups prevents any Tr i n t e r a c t i o n between the benzene TT system and the h i g h l y conjugated porphyrin system; thus the sample remains v i o l e t . In the d i a c i d of tetraphenylporphine, the porphinato core i s even more h i g h l y d i s t o r t e d due to van der Waals and Columbic r e p u l s i o n s of the four inner hydrogen atoms. In con t r a s t to the porphyrin f r e e base, the t i l t e d porphine skeleton allows the phenyl groups to r o t a t e toward the mean porphyrin plane making a d i h e d r a l angle of 21° with i t (Figure B.3). In t h i s case, strong ir i n t e r a c t i o n between the porphyrin and phenyl IT e l e c t r o n s gives r i s e to the green c o l o r of the d i a c i d species. - 1 6 8 -Figure B.3. Geometries for porphyrin diacids. -169-APPENDIX C THE EFFECT OF EXCITATION OF THE ELECTRON SHELL ON ANGULAR CORRELATION During r a d i o a c t i v e decay such as through a y-y cascade, there i s a p o s s i b i l i t y that a " h o l e " i s created i n one of the inner e l e c t r o n (K) s h e l l s , w i t h a r e s u l t i n g atomic s h e l l i n an e x c i t e d s t a t e . A f t e r the formation of the K-hole, the atomic s h e l l and nucleus tend to r e t u r n to t h e i r ground s t a t e s through y-y cascade and emission of Auger e l e c t r o n s and X-rays. F o l l o w i n g the Auger process, the atom can be charged. I t has been shown experimentally (161-164) that Cd can acquire an average charge 7e. The e l e c t r i c f i e l d o r i g i n a t i n g from these e x c i t e d atomic s h e l l s w i l l perturb the angular c o r r e l a t i o n . The p e r t u r b a t i o n depends on the mean l i f e of the e x i c t e d s t a t e ( x ^ ) and on the t r a n s i t i o n p r o b a b i l i t i e s f o r X-ray and Auger emission i n v a r i o u s -12 s h e l l s . This l i f e t i m e i s known to be very short (about 10 sec) f o r metals. In the case of i n s u l a t o r s i t may be as long as seconds. The i n t e r a c t i o n s t r e n g t h i s of the order of 500 MHz which means that there -9 w i l l be an i n f l u e n c e i f T * 1 0 sec or longer. The T„ f o r the 247-KeV N N e x c i t e d s t a t e of Cd^^" i s 1.25 x 10 ^sec, and a p e r t u r b a t i o n on angular c o r r e l a t i o n i s expected. No d e t a i l e d a n a l y s i s of t h i s e f f e c t has yet been developed. A f t e r the e l e c t r o n capture, the K-hole created can progress r a p i d l y towards the outermost s h e l l and i n the process generates more holes which a l s o move outwards during such m i g r a t i o n . This movement i s the consequence of Auger e f f e c t . Once these holes have reached the outermost s h e l l , they can decay only s l o w l y . I f t h e i r mean l i f e t i m e - 1 7 0 -i s long enough, a very strong p e r t u r b a t i o n on the angular c o r r e l a t i o n o f t e n r e s u l t e d . I f the p e r t u r b a t i o n i s of s u f f i c i e n t magnitude, the c o r r e l a t i o n can be attenuated below the hard core value (minimum v a l u e ) . The a t t e n t u a t i o n depends on the surrounding atomic environment which can be an i n s u l a t o r or a conductor. In the case i n which the r a d i o a c t i v e atom i s incorporated i n a substance of high c o n d u c t i v i t y ( i . e . the presence of conduction band), the e l e c t r o n s h e l l e x c i t a t i o n f o l l o w i n g the K-^capture remains f o r a -12 short period ( l e s s than 10 s e c ) . The nucleus i s thus remained un-perturbed by the e l e c t r i c f i e l d s a r i s i n g from such e x c i t e d s h e l l s . The presence of the conduction band allows r a p i d f r e e flow of e l e c t r o n s to " f i l l " the h o l e s , and thus leaves the r a d i o a c t i v e atom i n i t s ground s t a t e . This holds true whether the conductor c a r r y i n g the radionucleus i s i n a s o l i d or l i q u i d medium. In c o n t r a s t , i f the r a d i o a c t i v e atom i s incorporated i n a s o l i d i n s u l a t o r , the absence of a conduction band i n the r a d i o i s o t o p e ' s environment leads to a long decay time of the holes i n the outermost —8 s h e l l (>>10 s e c ) . Consequently, the i n s u l a t o r source should be expected to be attentuated. However, the s i t u a t i o n i s more complex when the i n s u l a t o r c a r r i e r i s i n s o l u t i o n . The i n f l u e n c e of the K-capture on the angular c o r r e l a t i o n s now depends on the r o t a t i o n a l c o r r e l a t i o n time (x ) and the i n t e r a c t i o n s between the i n s u l a t o r c molecules i n s o l u t i o n . I f the moelcule c a r r y i n g the decaying nucleus r o t a t e s very f a s t ( i . e . , i f the i n t e r a c t i o n i s strong and the r o t a t i o n a l c o r r e l a t i o n time i s s h o r t ) , the angular c o r r e l a t i o n w i l l be unperturbed, even i f the atomic s h e l l i s not i n i t s ground s t a t e . In the event when - 1 7 1 -the r o t a t i o n a l c o r r e l a t i o n time i s increased to a value much greater than ( i . e . , t C > > T J J ) » a p e r t u r b a t i o n on the angular c o r r e l a t i o n w i l l occur. -172-References A. D. Adler, J. Poly. Sci., Part C, 29, 73 (1970). J. P. Macquet and.T. Theophanides, Can. J. Chem., 51, 219 (1972). E. B. Fleischer and M. Krishamurthy, J. Am. Chem. Soc, 94, 1382 (1972). J. E. Falk, "Porphyrins and Metalloporphyrins", Elsevier, New York, 1964. "Structure Reports", Vol. 1-42, J. Trotter, Ed., Bohn, Scheltema & Holkema, Utrecht, 1978. M. F. Perutz, Scientific American, ^39, 92 (1978). T. K. Lejpert, J. D. Baldeschwieler, and D. A. Shirely, Nature (London), 220, 907 (1968). C. F. Meares, R. G. Bryant, J. D. Baldeschwieler, and D. A. Shirely, Proc. Natl. Acad. Sci. U.S.A., 6h_, 1155 (1969). D. A. Shirely, J. Chem. Phys., 55, 1512 (1971) C. F. Meares and D. G. Westmoreland, Proc. Cold Spring Harbor Sym. Quant. B i o l . , 36, 511 (1971). D. A. Shirley, J. Chem. Phys., _53, 465 (1970). J. C. Glass and G. Graf, Nature (London), 226, 635 (1970). A. G. Marshall and C. F. Meares, J. Chem. Phys., 56, 1 2 2 6 (1972). A. G. Marshall, L. G. Webelow, and C. F. Meares, J. Chem. Phys. 57, 364 (1972). J. L. Hoard, Science, 174, 1295 (1971). M. J. Hamor, T. A. Hamor, and J. L. Hoard, J. Am. Chem. Soc, 86, 6689 (1965). - 1 7 3 -17. M. J. Hamor, T. A. Hamor, and J. L. Hoard, J. Am. Chem. S o c , 86, 1938 (1964). 18. D. M. C o l l i n s , W. R. Scheidt, and J . L. Hoard, J . Am. Chem. S o c , 94, 6689 (1972). 19. R. J . P. W i l l i a m s , Fed. P r o c , Fed. Amer. Soc. Exp. B i o l . , 20, (Suppl. 10), 5 (1961). 20. M. D. G l i c k , G. H. Cohen, and J . L. Hoard, J . Am. Chem. S o c , 89, 1996 (1967). 21. D. M. C o l l i n s and J . L. Hoard, J . Am. Chem. S o c , 9_2, 3761 (1970). 22. J . L. Hoard, i n " S t r u c t u r a l Chemistry and Molecular B i o l o g y " , A. Rich and N. Dadvison, Eds., W. H. Freeman, San F r a n c i s o , C a l i f o r n i a , 1968, p537. 23. J . L. Hoard, M. J . Hamor, T. A. Hamor, and W. S. Caughey, J . Am. Chem. S o c , 87_, 2312 (1965). 24. W. A. Hendrickson and W. E. Love, Cold Spring Harbor Sym. Quant. B i o l . , 36, 1971. 25. R. Huber, 0. Epp, and H. Formanek, J . Mol. B i o l . , 52., 349 (1970). 26. S. E. V. P h i l l i p s , Nature (London), 27_1> 2 4 7 (1978). 27. J . L. Hoard, i n "Hemes and Hemoproteins", B. Chance, R. W. Estabrook, and T. Yonetani, Eds., Academic Press, New York, 1966, P 9 . 28. M. F. Pe r u t z , Nature (London), 228. 7 2 6 (1970). 228, 734 (1970) 29. M. F. P e r u t z , and L. F. TenEyck, Cold Spring Harbor Symp. Quant. B i o l . , 36, 295 (1971). 30. D. M. C o l l i n s , R. Countryman, and J . L. Hoard, J . Am. Chem. S o c , 94, 2066 (1972). - 1 7 4 -31. R. J . Radonovich, A. Bloon, and J . L. Hoard, J . Am. Chem. Soc.,. 94, 2073 (1972). 32. M. J . Hamor, T. A. Hamor, and J . L. Hoard, J . Am. Chem. S o c , 86, 1938 (1964). 33. E. B. F l e i s c h e r , C. K. M i l l e r , and L. E. Webb, J . Am. Chem. Soc., 86, 2343 (1964). 34. J . L. Hoard, Ann. N. Y. Acad. S c i . , 206, 18 (1973). 35. D. L. C u l l e n and E. F. Meyer, J r . , J . Am. Chem. S o c , 96, 2095 (1974). 36. J . A. Kaduk and W. R. Scheidt, Inorganic Chem., 13, 2095 (1974). 37. E. I . Meyer, J r . , Acta. C r y s t a l l o g r . , Sect. B. , _28, 2162 (1972). 38. E. B. F l e i s c h e r , J . Am. Chem. S o c , 85, 146 (1963). 39. T. A. Hamor, W. S. Caughey, and J . L. Hoard, J . Am. Chem. S o c , 87, 2305 (1965). 40. J . L. Hoard, G. H. Cohen, and M. D. G l i c k , J . Am. Chem. S o c , 89, 1992 (1967). 41. D. F. Koenig, Acta. C r y s t . , 18, 663 (1965). 42. L. E. Webb and E. B. F l e i s c h e r , J . Am. Chem. S o c , 87, 667 (1965). 43. S. S i l v e r s and A. T u l i n s k y , J . Am. Chem. S o c , 86, 927 (1964). 44. J . C. Kendrew, R. E. Dickerson, B. E. Strandberg, R. G. Hart, D. R. Davis, D. C. P h i l l i p s , and V. C. Shore, Nature, 185, 422 (1960). 45. D. A. Buckingham, J . P. Collman, J . L. Hoard, G. Lang, L. J . Randonovich, and C. A. Reed, c i t e d i n r e f . 47. 46. K. Adams, P. G. Rasmussen, K. Hatano, and W. R. Scheidt, c i t e d i n r e f . 47. -175-. 47. W. R. Schedit, i n "Porphyrins", D. Dolphin, Ed., Academic Pr e s s , New York, V o l . I l l , 1978, p463. 48. A. B. Hoffman, D. M. C o l l i n s , V. W. Day, E. B. F l e i s c h e r , T. S. S r i v a s t a v a , and J . L. Hoard, J . Am. Chem. S o c , 94, 3620 (1974). 49. A. Bloon and J . L. Hoard, c i t e d i n r e f . 47. 50. W. R. Scheidt and J . L. Hoard, (unpublished r e s u l t s ) , c i t e d i n r e f . 47. 51. L. J . Randonovich, W. S. Caughey, and J . L. Hoard, c i t e d i n r e f . 47. 52. J . E. Kenny, J . W. Buchler, and W. R. Scheidt, c i t e d i n r e f . 47. 53. W. R. Scheidt, J . A. Cunningham, and J . L. Hoard, J . Am. Chem. S o c , 95, 8299 (1973). 54. W. R. Scheidt, J . Am. Chem. S o c , 96, 90 (1974). 55. R. G. L i t t l e and J . A. I b e r s , J . Am. Chem. S o c , 9j>, 4452 (1974). 56. W. R. Scheidt and J . Ramanjua, Inorg. Chem., 14_, 2643 (1975). 57. P. N. Dwyer, P. Madura, and W. R. Scheidt, J . Am. Chem. S o c , _96, 4815 (1974). 58. M. F. Perutz, Nature (London), 237, 495 (1972). 59. H. Muirhead, J . M. Cox, L. M a z z a r e l l a and M. F. Perutz, J . Mol. B i o l . , 28, 117 (1967). 60. M. F. P e r u t z , H. Muirhead, J . M. Cox, L. C. G. Godman, F. S. Mathews, E. L. McGandy, and L. E. Webb, Nature (London), 219, 29 (1968). 61. M. F. Pe r u t z , H. Muirhead, J . M. Cox, and L. C. G. Godman, Nature (London), 217, pl31 (1968). - 1 7 6 -62. W. Bolton and M. F . Perutz , Nature (London) 228. 551 (1970). 63. H. Muirhead and J . Greer , Nature (London), 228, 516 (1970). 64. J . L . Hoard and W. R. Scheidt , Proc . N a t l . Acad. S c i . , U . S . A . , 70, 3919 (1973). 65. J . L . Hoard and W. R. Sche idt , Proc . N a t l . Acad. S c i . , U . S . A . , 71, 1578 (1974). 66. B. Gonzales, J . Kouba, S. Yee, C. A. Reed, J . F . K i r n e r , and W. R. Scheidt , J . Am. Chem. S o c , 3247 (1975). 67. J . J . H o p f i e l d , J . M o l . B i o l . , _7_7» 2 0 7 (1973). 68. M. F . Perutz , J . E . Ladner , S. R. Simon, and C. H . Ho, J . Mol . B i o l . , c i t e d i n r e f . 55. 69. M. F . Perutz , A . R. Roberts , S. R. Simon, and G. C. K. Roberts , J . B i o l . Chem., c i t e d i n r e f . 55. 70. M. F . Perutz , E . J . Heidner, J . E . Ladner, J . G. Bet t l e s tone , C. and E . F . Slade, J . B i o l . Chem., c i t e d in r e f . 55. 71. T. R. Lindstrom, C. Ho, and A. V. P i s c i o t t a , Nature (London), New B i o l . , 237, 263 (1972). 72. S. Ogawa, R. G. Shulman, and T . Yamane, J . Mol . B i o l . , 7_0, 291 (1972). 73. S. Ogawa and R. G. Shulman, J . Mol . B i o l . , _7J). 315 (1972). 74. G. Fermi , J . Mol . B i o l . , 97, 237 (1975). 75(a) F . Basolo , B. M. Hoffman, and J . A. Ibers , A c c Chem. Res . , 8^, 384 (1975). (b) B. M. Hoffman, J . Am. Chem. S o c , 97, 1688 (1975). 76. J . Leonard, T. Yonetan i , and J . B. C a l l i s , B iochemis try , 13. 1460 (1974). 77. T . L . Fabry , C. Simo, and K. Javaher ian , Biochim. B iophys ic s . A c t a . , 160, 118 (1968). -177-78. T. Yonetani, H. R. D r o t t , J . S. Lei g h , J r . , G. H. Reed, M. R. Waterman, and T. Asakura, J . B i o l . Chem., 245, 2998 (1970). 79. K. Moffat, R. S. Loe, and B. M. Hoffman, J . Am. Chem. S o c , 96, 5259 (1974). 80(a) Q. H. Gibson, B. M. Hoffman, R. H. Crepeau, S. J . E d e l s t e i n , and C. B u l l , Biochem. Biophys. Res. Commun., _59, 146 (1974). 80(b) C. B u l l , R. G. F i s h e r , and B. M. Hoffman, Biochem. Biophys. Res. Commun., 59, 140 (1974). 81(a) B. M. Hoffman, Q. H. Gibson, C. B u l l , R. H. Crepeau, S. J . E d e l s t e i n , R. G. F i s h e r , and M. J . McDonald, Ann. N. Y. Acad. S c i . , 244, 174 (1975). 81(b) S. J . E d e l s t e i n and Q. H. Gibson, J . B i o l . Chem., 250, 961 (1975). 250, 965 (1975). 82. J . E. O'Hagen, "Haematin Enzymes", Tergamon Press, Oxford, 1961, pl73. 83. B. M. Hoffman, C. A. S p i l b u r g , and D. H. P e t e r i n g , Cold Spring Harbor. Symp. Quant. B i o l . , 36, 343 (1971). 84. B. M. Hoffman and D. H. P e t e r i n g , P r o c N a t l . Acad. S c i . , U.S.A., 67, 637 (1970). 85. G. C. Hsu, C. A. S p i l b u r g , C. B u l l , and B. M. Hoffman, Proc. N a t l . Acad. S c i . , U.S.A., 69, 2122 (1972). 86. C. A. S p i l b u r g , B. M. Hoffman, and P. H. P e t e r i n g , J . B i o l . Chem., 247, 4219 (1972). 87. W. H. Woodruff, T. G. S p i r o , and T. Yonetani, Proc. N a t l . Acad. S c i . , U.S.A., 71, 1065 (1974). -178-88. R. G. L i t t l e , B. M. Hoffman, and J . A. I b e r s , B i o i n o r g . Chem., 3, 207 (1974). 89. J . C. Kendrew, Science, 139, 1259 (1963). 90. J . C. Kendrew, Brookhav. Sym. B i o l . , L5, 216 (1962). 91. M. F. Pe r u t z , M. G. Rossman, A. F. C u l l i s , H. Muirhead, G. W i l l , and C. T. North, Nature (London) 185, 416 (1960). 92. A. F. C u l l i s , H. Muirhead, M. F. Perutz, M. G. Rossman, and A. C. T. North, Proc. Roy. S o c , (Lond.), Ser. A., 263, 161 (1962). 93. J . C. Kendrew, H. C. Watson, B. E. Strandberg, R. E. Dickerson, D. C. P h i l l i p s , and V. C. Shore, Nature (London), 190, 666 (1961). 94. M. F. Pe r u t z , " P r o t e i n s and N u c l e i A c i d s " , Holland, E l s e v i e r P u b l . , 1962. 95. T. Takano, J . Mol. B i o l . , 110, 537 (1977). 96. L. F. Ten Eyck and Arnone, J . Mol. B i o l . , 100, 3 (1976). 97. R. E. Dickerson and I. Geis, "The Str u c t u r e and A c t i o n of P r o t e i n s " , W. A. Benjamin, I n c , C a l i f o r n i a , 1969. 98. B. R. James, i n "Porphyrins", D. Dolphin, Ed., Academic Press, Inc., New York, V o l . 5, 1978, p205. 99. D. Dolphin, "Porphyrins", Academic P r e s s , Inc., New York, 1978. 100. J . P. Collman, R. R. Gagne, C. A. Reed, W. T. Robinson, and G. A. Rodley, P r o c N a t l . Acad. S c i . , 71, 1326 (1974). 101. H. Diekmann, C. K. Chang, and T. G. T r a y l o r , J . Am. Chem. S o c , 93, 4068 (1971). 102. J . Almog, J . E. Baldwin, R. L. Dyer, and M. P e t e r s , J . Am. Chem. S o c , 9J_, 227 (1975). -179-103. J . P. Collman, R. R. Gagne, T. R. H a l b e r t , J . C. Marchon, and C. A. Reed, J . Am. Chem. S o c , 95, 7868 (1973). 104. J . P. Collman, R. R. Gagne, H. B. Gray, and J . W. Hare, J . Am. Chem. S o c , 96, 6522 (1974). 105. J . P. Collman and W. T. Robinson, Nature, 23,5, 438 (1972). 106(a)C. K. Chang and T. G. T a y l o r , Proc. N a t l . Acad. S c i . , U.S.A., 70, 2647 (1973). ( b ) I b i d , J . Am. Chem. S o c , 95, 5810 (1973). 107. M. B h a t t i , W. B h a t t i , and E. Mast, Inorg. Nucl. Chem. L e t t e r s , 8, 133 (1972). 108. The computer programs used i n c l u d e l o c a l l y w r i t t e n programs f o r data processing and l o c a l l y modified v e r s i o n s of the f o l l o w i n g : 0RFLS f u l l m atrix l e a s t squares and ORFFE, f u n c t i o n and e r r o r s by W. R. Busing, K. 0. M a r t i n and H. A. Levy; P a t t e r s o n and Fo u r i e r s y n t h e s i s , FORDAP by A. Z a l k i n ; c r y s t a l s t r u c t u r e i l l u s t r a t i o n s , ORTEP I I by C. K. Johnson. 109. " I n t e r n a t i o n a l Tables f o r X-ray C r y s t a l l o g r a p h y " , V o l . IV, Kynoch Press, Birmingham, England, 1972, p.99. 110. D. T. Cromer and D. Liberman, J . Chem. Phys. 53, 1891 (1970). 111. T. Mashiko, M. E. Kastner, K. S p a r t a l i a n , W. R. Scheidt, and C. A. Reed, J . Am. Chem. S o c , 100, 6354 (1978). 112. W. H. Huestic and M. A. R a f t e r y , Biochemistry, 11, 1648 (1972). 113. E. J . Heidner, R. C. Ladner, and M. F. Pe r u t z , J . Mol. B i o l . , 104, 707 (1976). 114. A. F i t z g e r a l d , p r i v a t e communication. 115. J . L. Hoard, N. Kim, and J . W. Buchler, A b s t r a c t s , 167th N a t i o n a l Meeting of the American Chemical S o c i e t y , Los Angeles, CA (1974), No. INOR 75. -180---116. D. C. C u l l e n , E. F. Meyer, J r . , and K. M. Smith, Inorg. Chem., 16, 1179 (1977). 117. F. Molinaro and J.- A. I b e r s , Inorg. Chem., 15, 2278 (1976). 118. G. B. Jameson, F. Molarino, J . A. I b e r s , J . P. Collman, J . I . Brauman, E. Rose, and K. S. S u s l i c k , J . Am. Chem. S o c , 100, 6769 (1978). 119. L. P. Spauling, E. G. E l l e r , J . A. Bertrand, and R. H. F e l t o n , J . Am. Chem. S o c , 96, 982 (1974). 120. D. L. C u l l e n and E. F. Meyer, J r . , Acta C r y s t a l l o g r . , Sect. B. , 32, 2259 (1976). 121. B. M. Chen, Ph.D. Thesis, Michigan State U n i v e r s i t y , 1970. 122. R. Timkovich and A. T u l i n s k y , J . Am. Chem. S o c , 91, 4430 (1969). 123. V. W. Day, B. R. S t u l t s , E. L. Tasset, R. S. M a r i a n e l l i , and L. J . Boucher, Inorg. Nucl. Chem. L e t t e r s , 11, 509 (1975). 124. J . P. Collman, J . I . Brauman, K. M. Doxsee, T. R. H a l b e r t , S. E. Hayes, and K. S. S u s l i c k , J . Am. Chem. S o c , 100, 2761 (1978). 125. S. S. Eaton and G. R. Eaton, J . Am. Chem. S o c , 99, 6594 (1977). 126. R. J . Abraham, G. H. Barnett, and K. M. Smith, J . Chem. S o c , P e r k i n I , 2142 (1973). 127. L. S t r y e r , J . Mol. B i o l . , 13, 482 (1965). 128. T. R. Stengle and J . D. Baldeschwieler, Proc. N a t l . Acad. S c i . , U.S.A., 55, 1020 (1966). 129. C. L. Hamilton and H. M. McConnell, i n " S t r u c t u r a l Chemistry and Molecular B i o l o g y " , A. Rich and N. Davidson, Eds., Freeman & Co., San F r a n c i s c o , 1968, p l l 5 . -181-130. H. Frauenfelder and R. M. S t e f f e n , i n "Alpha-, Beta-, and Gamma-ray Spectroscopy", K. Siegbahn, Ed., North-Holland, Amsterdam, 1965, p997. 131. R. M. S t e f f e n , Phys. Rev., 103, 116 (1956). 132. "Table of Isotopes", C. M. Lederer, J . M. Hollander, and J . Perlman, Eds., John Wiley & Son, New York, 1968, p252. 133. A. Abragam and R. V. Pound, Phys. Rev., 92_, 943 (1953). 134. M. Blume, i n "Hyperfine S t r u c t u r e and Nuclear R a d i a t i o n s " , E. Matthias and D. A. S h i r l e y , Eds., North-Holland, Amsterdam, 1968, p991. 135. P. Debye, " P o l a r Molecules", Dover P u b l i c a t i o n s , New York, 1965. 136. N. Bloembergen, E. M. P u r c e l l , and R. V. Pound, Phys. Rev., 73, 679 (1948). 137. P. B. Hemmig and R. M. S t e f f e n , Phys. Rev., 92, 832 (1953). 138. P. Lehmann and J . M i l l e r , J . Phys. Radium, 17_, 526 (1956). 139. J . S. B a r r e t t , J . A. Cameron, P. R. Gardner, L. K e z t h e l y i , W. V. P r e s t w i c h , and M. Kaplan, J . Chem. Phys., 53, 759 (1970) 140. R. M. Lynden-Bell, Mol. Phys., 21, 891 (1971). 141. E. M a t t h i a s , B. Olsen, and W. Schneider, A r k i v For F y s i k , 24, 245 (1963). 142. E. M a t t h i a s , W. Schneider, and R. M. S t e f f e n , Phys. L e t t s . , 4, 41 (1963). 143. E. M a t t h i a s , W. Schneider, and R. M. S t e f f e n , A r k i v For F y s i k , 24, 97 (1963). 144. R. W. Sommerfeldt and L. Schecter, Phys. L e t t s . , 3, 5 (1962). -182-145. D. A. Goodwin, C. F. Meares, and C. H. Song, Radiology, 105, 699 (1972). 146. H. S. Stern , D. A. Goodwin, and W. S c h e f f e l , et a l . , Nucleonics, _25, 62 (19C7). 147. F. Hosain, P. A. Mcl n t y r e , K. Poulose, et a l . , C l i n . Chim. Acta., 24, 69 (1969). 148. P. W. M a r t i n and C. A. K a l f a s , Chem. Phys. L e t t s . , _5_7, 2 7 9 (1978). 149. F. F i s c h e r and H. Bock, Z. P h y s i o l . Chem., 25_5, 1 (1938). 150. J . F. T a y l o r , J . B i o l . Chem., 135, 569 (1940). 151. E. Breslow, J . B i o l . Chem., _239, 486 (1964). 152. T. S. S r i v a s t a v a , Biochim. Biophys. A c t a . , 491, 599 (1977). 153. F. James and M. Ross, Comput. Phys. Commun., 1_0, 343 (1975). 154. J . Yguerabide, i n "Methods i n Enzymology", C. H. W. H i r s and S. N. Timasheff, Eds., Academic Press, New York, 1972, V o l . 26, part C, p.528. 155. "Hand-Book of Biochemistry - Selected Data f o r Molecular B i o l o g y " , H. A. Sober, Ed., The Chemical Rubber Co., Ohio, 1970, pClO. 156. D. D. Haas, R. V. Mustacich, B. A. Smith, and B. R. Ware, Biochem. Biophys. Res. Commun., 59., 174 (1974). 157. C. R. Jones and C. S. Johnson, J r . , J . Chem. Phys., j55_, 2020 (1976). 158. D. D. Haas and B. R. Ware, Biochemistry, 1_7, 4946 (1978). 159. E. J . Shimshick and H. M. McConnell, Biochem. Biophys. Res. Commun., 46, 1 (1972). 160. R. C. McCallery, E. J . Shimshick, and H. M. McConnell, Chem. Phys. L e t t s . , 13, 115 (1972). -183-161. M. L. Perlman and J . A. M i s k e l , Phys. Rev., 91, 899 (1953). 162. S. Wexler, Phys. Rev., 93, 182 (1954). 163. 0. Kofoed-Hansen, Phys. Rev., 96, 1045 (1954). 164. P. H. S n e l l and F. Pleasonton, Phys. Rev., 102, 1419 (1956). 

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